Thermostable neutral metalloproteases

ABSTRACT

The present invention provides methods and compositions comprising at least one neutral metalloprotease enzyme that has improved storage stability. In some embodiments, the neutral metalloprotease finds use in cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions comprising neutral metalloprotease(s) obtained from  Bacillus  sp. In some more particularly preferred embodiments, the neutral metalloprotease is obtained from  B. amyloliquefaciens . In still further preferred embodiments, the neutral metalloprotease is a variant of the  B. amyloliquefaciens  neutral metalloprotease. In yet additional embodiments, the neutral metalloprotease is a homolog of the  B. amyloliquefaciens  neutral metalloprotease. The present invention finds particular use in applications including, but not limited to cleaning, bleaching and disinfecting.

The present application is a continuation of U.S. patent applicationSer. No. 11/581,102, filed on Oct. 12, 2006, now abandoned which claimspriority to U.S. Provisional patent application Ser. No. 60/726,448,filed Oct. 12, 2005, which are herein incorporated in their entirety.

FIELD OF THE INVENTION

The present invention provides methods and compositions comprising atleast one neutral metalloprotease enzyme that has improved storagestability. In some embodiments, the neutral metalloprotease finds use incleaning and other applications. In some particularly preferredembodiments, the present invention provides methods and compositionscomprising neutral metalloprotease(s) obtained from Bacillus sp. In somemore particularly preferred embodiments, the neutral metalloprotease isobtained from B. amyloliquefaciens. In still further preferredembodiments, the neutral metalloprotease is a variant of the B.amyloliquefaciens neutral metalloprotease. In yet additionalembodiments, the neutral metalloprotease is a homolog of the B.amyloliquefaciens neutral metalloprotease. The present invention findsparticular use in applications including, but not limited to cleaning,bleaching and disinfecting.

BACKGROUND OF THE INVENTION

Detergent and other cleaning compositions typically include a complexcombination of active ingredients. For example, most cleaning productsinclude a surfactant system, enzymes for cleaning, bleaching agents,builders, suds suppressors, soil-suspending agents, soil-release agents,optical brighteners, softening agents, dispersants, dye transferinhibition compounds, abrasives, bactericides, and perfumes. Despite thecomplexity of current detergents, there are many stains that aredifficult to completely remove. Furthermore, there is often residuebuild-up, which results in discoloration (e.g., yellowing) anddiminished aesthetics due to incomplete cleaning. These problems arecompounded by the increased use of low (e.g., cold water) washtemperatures and shorter washing cycles. Moreover, many stains arecomposed of complex mixtures of fibrous material, mainly incorporatingcarbohydrates and carbohydrate derivatives, fiber, and cell wallcomponents (e.g., plant material, wood, mud/clay based soil, and fruit).These stains present difficult challenges to the formulation and use ofcleaning compositions.

In addition, colored garments tend to wear and show appearance losses. Aportion of this color loss is due to abrasion in the laundering process,particularly in automated washing and drying machines. Moreover, tensilestrength loss of fabric appears to be an unavoidable result ofmechanical and chemical action due to use, wearing, and/or washing anddrying. Thus, a means to efficiently and effectively wash coloredgarments so that these appearance losses are minimized is needed.

In sum, despite improvements in the capabilities of cleaningcompositions, there remains a need in the art for detergents that removestains, maintain fabric color and appearance, and prevent dye transfer.In addition, there remains a need for detergent and/or fabric carecompositions that provide and/or restore tensile strength, as well asprovide anti-wrinkle, anti-bobbling, and/or anti-shrinkage properties tofabrics, as well as provide static control, fabric softness, maintainthe desired color appearance, and fabric anti-wear properties andbenefits. In particular, there remains a need for the inclusion ofcompositions that are capable of removing the colored components ofstains, which often remain attached to the fabric being laundered. Inaddition, there remains a need for improved methods and compositionssuitable for textile bleaching.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions comprising atleast one neutral metalloprotease enzyme that has improved storagestability. In some embodiments, the neutral metalloprotease finds use incleaning and other applications. In some particularly preferredembodiments, the present invention provides methods and compositionscomprising neutral metalloprotease(s) obtained from Bacillus sp. In somemore particularly preferred embodiments, the neutral metalloprotease isobtained from B. amyloliquefaciens. In still further preferredembodiments, the neutral metalloprotease is a variant of the B.amyloliquefaciens neutral metalloprotease. In yet additionalembodiments, the neutral metalloprotease is a homolog of the B.amyloliquefaciens neutral metalloprotease. The present invention findsparticular use in applications including, but not limited to cleaning,bleaching and disinfecting.

The present invention provides novel neutral metalloproteases, novelgenetic material encoding the neutral metalloprotease enzymes, andneutral metalloprotease proteins obtained from Bacillus sp., inparticular, B. amyloliquefaciens, and variant proteins developedtherefrom. In particular, the present invention provides neutralmetalloprotease compositions obtained from Bacillus sp., particularly B.amyloliquefaciens, DNA encoding the protease, vectors comprising the DNAencoding the neutral metalloprotease, host cells transformed with thevector DNA, and an enzyme produced by the host cells. The presentinvention also provides cleaning compositions (e.g., detergentcompositions), animal feed compositions, and textile and leatherprocessing compositions comprising neutral metalloprotease(s) obtainedfrom a Bacillus species, in particular, B. amyloliquefaciens. Inalternative embodiments, the present invention provides mutant (i.e.,variant) neutral metalloproteases derived from the wild-type neutralmetalloproteases described herein. These mutant neutral metalloproteasesalso find use in numerous applications.

The present invention provides isolated neutral metalloproteasesobtained from a Bacillus species, in particular, B. amyloliquefaciens.In further embodiments, the neutral metalloprotease comprises the aminoacid sequence set forth in SEQ ID NO:3. In additional embodiments, thepresent invention provides isolated neutral metalloproteases comprisingat least 45% amino acid identity with the neutral metalloproteasecomprising SEQ ID NO:3. In some embodiments, the isolated neutralmetalloproteases comprise at least 50% identity, preferably at least55%, more preferably at least 60%, yet more preferably at least 65%,even more preferably at least 70%, more preferably at least 75%, stillmore preferably at least 80%, more preferably 85%, yet more preferably90%, even more preferably at least 95%, and most preferably 99% identitywith the neutral metalloprotease comprising SEQ ID NO:3.

The present invention also provides isolated neutral metalloproteaseshaving immunological cross-reactivity with the metalloprotease obtainedfrom B. amyloliquefaciens, as well as compositions comprising theseneutral metalloproteases. In alternative embodiments, the neutralmetalloproteases have immunological cross-reactivity with neutralmetalloproteases comprising the amino acid sequence set forth in SEQ IDNO:3 and/or SEQ ID NO:18. In still further embodiments, the neutralmetalloproteases have cross-reactivity with fragments (i.e., portions)of the neutral metalloprotease of B. amyloliquefaciens, and/or neutralmetalloprotease comprising the amino acid sequence set forth in SEQ IDNO:3 and/or SEQ ID NO:18. Indeed, it is intended that the presentinvention encompass fragments (e.g., epitopes) of the B.amyloliquefaciens metalloprotease that stimulate an immune response inanimals (including, but not limited to humans) and/or are recognized byantibodies of any class. The present invention further encompassesepitopes on metalloproteases that are cross-reactive with B.amyloliquefaciens metalloprotease epitopes. In some embodiments, themetalloprotease epitopes are recognized by antibodies, but do notstimulate an immune response in animals (including, but not limited tohumans), while in other embodiments, the metalloprotease epitopesstimulate an immune response in at least one animal species (including,but not limited to humans) and are recognized by antibodies of anyclass. The present invention also provides means and compositions foridentifying and assessing cross-reactive epitopes.

In some embodiments, the present invention provides the amino acidsequences set forth in SEQ ID NO:3 and/or SEQ ID NO:4. In alternativeembodiments, the sequence comprises substitutions at least one aminoacid position in SEQ ID NO:3 and/or SEQ ID NO:4. In some preferredembodiments, the present invention provides neutral metalloproteasevariants having an amino acid sequence comprising at least onesubstitution of an amino acid made at a position equivalent to aposition in a B. amyloliquefaciens neutral metalloprotease comprisingthe amino acid sequence set forth in SEQ ID NO:3 and/or SEQ ID NO:4. Inalternative embodiments, the present invention provides neutralmetalloprotease variants having an amino acid sequence comprising atleast one substitution of an amino acid made at a position equivalent toa position in a B. amyloliquefaciens neutral metalloprotease comprisingat least a portion of SEQ ID NO:3 and/or SEQ ID NO:4. In somealternative preferred embodiments, the neutral metalloproteases comprisemultiple mutations in at least a portion of SEQ ID NO:3, 4 and/or 18.

In yet additional embodiments, the present invention provides the aminoacid sequence set forth in SEQ ID NO:13. In alternative embodiments, thesequence comprises substitutions at least one amino acid position in SEQID NO:13. In some preferred embodiments, the present invention providesneutral metalloprotease variants having an amino acid sequencecomprising at least one substitution of an amino acid made at a positionequivalent to a position in a B. amyloliquefaciens neutralmetalloprotease comprising the amino acid sequence set forth in SEQ IDNO:13. In alternative embodiments, the present invention providesneutral metalloprotease variants having an amino acid sequencecomprising at least one substitution of an amino acid made at a positionequivalent to a position in a B. amyloliquefaciens neutralmetalloprotease comprising at least a portion of SEQ ID NO:13. In somealternative preferred embodiments, the neutral metalloproteases comprisemultiple mutations in at least a portion of SEQ ID NO:13.

In some particularly preferred embodiments, these variants have improvedperformance as compared to wild-type B. amyloliquefaciens neutralmetalloprotease. The present invention also provides neutralmetalloprotease variants having at least one improved property ascompared to the wild-type neutral metalloprotease. In some additionalparticularly preferred embodiments, these variants have improvedstability as compared to wild-type B. amyloliquefaciens neutralmetalloprotease. In some further preferred embodiments, these variantshave improved thermostability as compared to wild-type B.amyloliquefaciens neutral metalloprotease. In yet additional preferredembodiments, these variants have improved performance under lower orhigher pH conditions, as compared to wild-type B. amyloliquefaciensneutral metalloprotease.

The present invention also provides neutral metalloproteases comprisingat least a portion of the amino acid sequence set forth in SEQ ID NO:3,4 and/or 18. In some embodiments, the nucleotide sequences encodingthese neutral metalloproteases comprise a nucleotide sequence selectedfrom SEQ ID NOS:1, 2, 12, and/or 13. In some embodiments, the neutralmetalloproteases are variants having amino acid sequences that aresimilar to that set forth in SEQ ID NO:3 and/or SEQ ID NO:4. In yetadditional embodiments, the neutral metalloproteases are variants and/orhomologs. In still further embodiments, the neutral metalloproteases arethose set forth in any of FIGS. 3 through 5. In other embodiments, theneutral metalloproteases are variants of those set forth in FIGS. 3, 4and/or 5.

The present invention also provides expression vectors comprising apolynucleotide sequence encoding at least a portion of the neutralmetalloprotease set forth in SEQ ID NO:3. The present invention furtherprovides expression vectors comprising a polynucleotide sequences thatencode at least one neutral metalloprotease variant having amino acidsequence(s) comprising at least one substitution of an amino acid madeat a position equivalent to a position in a Bacillus neutralmetalloprotease comprising the amino acid sequence set forth in SEQ IDNO:3. In further embodiments, the present invention provides host cellscomprising these expression vectors. In some particularly preferredembodiments, the host cells are selected from the group consisting ofBacillus sp. The present invention also provides the neutralmetalloproteases produced by the host cells.

The present invention also provides compositions comprising at least aportion of an isolated neutral metalloprotease of obtained from aBacillus sp., particularly, B. amyloliquefaciens, wherein at least aportion of the neutral metalloprotease is encoded by a polynucleotidesequence selected from SEQ ID NOS:1, 2, 12 and/or 13. In furtherembodiments, the present invention provides host cells comprising theseexpression vectors. In some particularly preferred embodiments, the hostcells are Bacillus sp. The present invention also provides the neutralmetalloproteases produced by the host cells.

The present invention also provides variant neutral metalloproteases,wherein the neutral metalloproteases comprise at least one substitutioncorresponding to the amino acid positions in SEQ ID NO:3 and/or SEQ IDNO:18, and wherein variant metalloproteases have better performance inat least one property, as compared to wild-type B. amyloliquefaciensmetalloprotease.

The present invention also provides variant amino acids, wherein thevariants comprise at least one substitution of an amino acid made at aposition equivalent to a position in a neutral metalloproteasecomprising the amino acid set forth in SEQ ID NO:18, wherein theposition(s) is or are selected from positions 1, 3, 4, 5, 6, 11, 12, 13,14, 16, 21, 23, 24, 25, 31, 32, 33, 35, 36, 38, 44, 45, 46, 47, 48, 49,50, 51, 54, 55, 58, 59, 60, 61, 62, 63, 65, 66, 69, 70, 76, 85, 86, 87,88, 90, 91, 92, 96, 97, 98, 99, 100, 102, 109, 110, 111, 112, 113, 115,117, 119, 127, 128, 129, 130, 132, 135, 136, 137, 138, 139, 140, 146,148, 151, 152, 153, 154, 155, 157, 158, 159, 161, 162, 169, 173, 178,179, 180, 181, 183, 184, 186, 190, 191, 192, 196, 198, 199, 200, 202,203, 204, 205, 210, 211, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 228, 229, 237, 239, 240, 243, 244, 245, 248, 252, 253, 260,261, 263, 264, 265, 267, 269, 270, 273, 277, 280, 282, 283, 284, 285,286, 288, 289, 290, 292, 293, 296, 297, and 299.

The present invention also provides isolated neutral metalloproteasevariants having an amino acid sequence comprising at least onesubstitution of an amino acid made at a position equivalent to aposition in a neutral metalloprotease comprising the amino acid sequenceset forth in SEQ ID NO:18. In some embodiments, the isolated neutralmetalloprotease variants have substitutions that are made at positionsequivalent to positions 1, 3, 4, 5, 6, 11, 12, 13, 14, 16, 21, 23, 24,25, 31, 32, 33, 35, 36, 38, 44, 45, 46, 47, 48, 49, 50, 51, 54, 55, 58,59, 60, 61, 62, 63, 65, 66, 69, 70, 76, 85, 86, 87, 88, 90, 91, 92, 96,97, 98, 99, 100, 102, 109, 110, 111, 112, 113, 115, 117, 119, 127, 128,129, 130, 132, 135, 136, 137, 138, 139, 140, 146, 148, 151, 152, 153,154, 155, 157, 158, 159, 161, 162, 169, 173, 178, 179, 180, 181, 183,184, 186, 190, 191, 192, 196, 198, 199, 200, 202, 203, 204, 205, 210,211, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 228, 229,237, 239, 240, 243, 244, 245, 248, 252, 253, 260, 261, 263, 264, 265,267, 269, 270, 273, 277, 280, 282, 283, 284, 285, 286, 288, 289, 290,292, 293, 296, 297, and 299 of a neutral metalloprotease comprising anamino acid sequence set forth in SEQ ID NO:18.

In additional embodiments, the isolated neutral metalloprotease variantcomprise comprises at least one mutation selected from T004C, T004E,T004H, T0041, T004K, T004L, T004M, T004N, T004P, T004R, T004S, T004V,T004W, T004Y, G012D, G012E, G0121, G012K, G012L, G012M, G012Q, G012R,G012T, G012V, G012W, K013A, K013C, K013D, K013E, K013F, K013G, K013H,K0131, K013L, K013M, K013N, K013Q, K013S, K013T, K013V, K013Y, T014F,T014G, T014H, T0141, T014K, T014L, T014M, T014P, T014Q, T014R, T014S,T014V, T014W, T014Y, S023A, S023D, S023F, S023G, S0231, S023K, S023L,S023M, S023N, S023P, S023Q, S023R, S023S, S023T, S023V, S023W, S023Y,G024A, G024D, G024F, G024G, G024H, G0241, G024K, G024L, G024M, G024N,G024P, G024R, G024S, G024T, G024V, G024W, G024Y, K033H, Q045C, Q045D,Q045E, Q045F, Q045H, Q0451, Q045K, Q045L, Q045M, Q045N, Q045P, Q045R,Q045T, Q045W, N046A, N046C, N046E, N046F, N046G, N046H, N0461, N046K,N046L, N046M, N046P, N046Q, N046R, N046S, N046T, N046V, N046W, N046Y,R047E, R047K, R047L, R047M, R47Q, R047S, R047T, Y049A, Y049C, Y049D,Y049E, Y049F, Y049H, Y0491, Y049K, Y049L, Y049N, Y049R, Y049S, Y049T,Y049V, Y049W, N050D, N050F, N050G, N050H, N0501, N050K, N050L, N050M,N050P, N050Q, N050R, N050W, N050Y, T054C, T054D, T054E, T054F, T054G,T054H, T054I T054K, T054L, T054M, T054N, T054P, T054Q, T054R, T054S,T054V, T054W, T054Y, S058D, S058H, S0581, S058L, S058N, S058P, S058Q,T059A, T059C, T059E, T059G, T059H, T0591, T059K, T059L T059M, T059N,T059P, T059Q, T059R, T059S, T059V, T059W, T060D, T060F, T0601, T060K,T060L, T060N, T060Q, T060R, T060V, T060W, T060Y, T065C, T065E, T065F,T065H, T065I, T065K, T065L, T065M, T065P, T065Q, T065R, T065V, T065Y,S066C, S066D, S066E, S066F, S066H, S0661, S066K, S066L, S066N, S066P,S066Q, S066R, S066T, S066V, S066W, S066Y, Q087A, Q087D, Q087E, Q087H,Q0871, Q087K, Q087L, Q087M, Q087N, Q087R, Q087S, Q087T, Q087V, Q087W,N090C, N090D, N090E, N090F, N090G, N090H, N090K, N090L, N090R, N090T,N096G, N096H, N096K, N096R, K097H, K097Q, K097W, K100A, K100D, K100E,K100F, K100H, K100N, K100P, K100Q, K100R, K100S, K100V, K100Y, R110A,R110C, R110E, R110H, R110K, R110L, R110M, R110N, R110Q, R110S, R110Y,D119E, D119H, D1191, D119L, D119Q, D119R, D119S, D119T, D119V, D119W,G128C, G128F, G128H, G128K, G128L, G128M, G128N, G128Q, G128R, G128W,G128Y, S129A, S129C, S129D, S129F, S129G, S129H, S129I, S129K, S129L,S129M, S129Q, S129R, S129T, S129V, S129W, S129Y, F1301, F130K, F130L,F130M, F130Q, F130R, F130T, F130V, F130Y, S135P, G136I, G136L, G136P,G136V, G136W, G136Y, S137A, M138I, M138K, M138L, M138Q, M138V, D139A,D139C, D139E, D139G, D139H, D139I, D139K, D139L, D139M, D139P, D139R,D139S, D139V, D139W, D139Y, V140C, Q151I, E152A, E152C, E152D, E152F,E152G, E152H, E152L, E152M, E152N, E152R, E152S, E152W, N155D, N155K,N155Q, N155R, D178A, D178C, D178G, D178H, D178K, D178L, D178M, D178N,D178P, D178Q, D178R, D178S, D178T, D178V, D178W, D178Y, T179A, T179F,T179H, T179I, T179K, T179L, T179M, T179N, T179P, T179Q, T179R, T179S,T179V, T179W, T179Y, E186A, E186C, E186D, E186G, E186H, E186K, E186L,E186M, E186N, E186P, E186Q, E186R, E186S, E186T, E186V, E186W, E186Y,V190H, V190I, V190K, V190L, V190Q, V190R, S191F, S191G, S191H, S191I,S191K, S191L, S191N, S191Q, S191R, S191W, L198M, L198V, S199C, S199D,S199E, S199F, S199I, S199K, S199L, S199N, S199Q, S199R, S199V, Y204H,Y204T, G205F, G205H, G205L, G205M, G205N, G205R, G205S, G205Y, K211A,K211C, K211D, K211G, K211M, K211N, K211Q, K211R, K211S, K211T, K211V,K214A, K214C, K214E, K2141, K214L, K214M, K214N, K214Q, K214R, K214S,K214V, L216A, L216C, L216F, L216H, L216Q, L216R, L216S, L216Y, N218K,N218P, T219D, D220A, D220E, D220H, D220K, D220N, D220P, A221D, A221E,A221F, A2211, A221K, A221L, A221M, A221N, A221S, A221V, A221Y, G222C,G222H, G222N, G222R, Y224F, Y224H, Y224N, Y224R, T243C, T243G, T243H,T2431, T243K, T243L, T243Q, T243R, T243W, T243Y, K244A, K244C, K244D,K244E, K244F, K244G, K244L, K244M, K244N, K244Q, K244S, K244T, K244V,K244W, K244Y, V260A, V260D, V260E, V260G, V260H, V2601, V260K, V260L,V260M, V260P, V260Q, V260R V260S, V260T, V260W, V260Y, Y261C, Y261F,Y2611, Y261L, T263E, T263F, T263H, T2631, T263L, T263M, T263Q, T263V,T263W, T263Y, S265A, S265C, S265D, S265E, S265K, S265N, S265P, S265Q,S265R, S265T, S265V, S265W, K269E, K269F, K269G, K269H, K2691, K269L,K269M, K269N, K269P, K269Q, K269S, K269T, K269V, K269W, K269Y, A273C,A273D, A273H, A2731, A273K, A273L, A273N, A273Q, A273R, A273Y, R280A,R280c, R280D, R280E, R280F, R280G, R280H, R280K, R280L, R280M, R280S,R280T, R280V, R280W, R280Y, L282F, L282G, L282H, L2821, L282K, L282M,L282N, L282Q, L282R, L282V, L282Y, S285A, S285C, S285D, S285E, S285K,S285P, S285Q, S285R, S285W, Q286A, Q286D, Q286E, Q286K, Q286P, Q286R,A289C, A289D, A289E, A289K, A289L, A289R, A293C, A293R, N296C, N296D,N296E, N296K, N296R, N296V, A297C, A297K, A297N, A297Q, A297R, andG299N.

In still further embodiments the present invention provides isolatedvariant neutral metalloproteases, wherein the metalloprotease comprisesmultiple mutations selected from S023W/G024M, T004V/S023W/G024W,S023W/G024Y/A288V, T004L/S023W/G024Y, N046Q/N050F/T054L,N050Y/T059R/S129Q, S023W/G024W, A273H/S285P/E292G, S023Y/G024Y,S023Y/G024W, T004S/S023Y/G024W, N046Q/T054K, S023W/G024Y, T004V/S023W,T059K/S066N, N046Q/N050W/T054H/T153A, T004V/S023W/G024Y,L282M/Q286P/A289R, N046Q/R047K/N050Y/T054K, L044Q/T263W/S285R,T004L/S023W/G024W, R047K/N050F/T054K, A273H/S285R, N050Y/T059K/S066Q,T054K/Q192K, N046Q/N050W, L282M/Q286K, T059K/S066Q, T004S/S023W,L282M/Q286R/A289R/K011N, L282M/A289R, N046Q/N050W/T054H, T059K/S129Q,T004S/S023N/G024Y/F210L, T004V/S023W/G024M/A289V,L282M/Q286K/A289R/S132T, N050W/T054H, L282M/Q286R, L282F/Q286K/A289R,T059R/S066Q, R047K/N050W/T054H, S265P/L282M/Q286K/A289R,L282M/Q286R/T229S, L282F/Q286K, T263W/S285R, S265P/L282M/Q286K,T263H/A273H/S285R, T059R/S129V, S032T/T263H/A273H/S285R,T059R/S066Q/S129Q, T004S/G024W, T004V/S023W/G024M, T059K/S066Q/S129Q,L282M/Q286K/A289R/1253V, T004V/S023Y/G024W, T059R/S066N/S129Q,N050F/T054L, T004S/S023N/G024W, T059R/S066N, T059R/S066N/S129V,Q286R/A289R, N046Q/R047K/N050F/T054K, S265P/L282M/Q286P/A289R,S265P/L282M/Q286R/A289R Q062K/S066Q/S129I, S023N/G024W,N046Q/R047K/N050W/T054H, R047K/T054K, T004L/G024W, T014M/T059R/S129V,T059R/S066Q/N092S/S129I, R047K/N050W/T054K, T004V/G024W,N047K/N050F/T054K, S265P/L282F/Q286K/N061Y, L282F/Q286K/E159V,T004V/S023Y/G024M, S265P/L282F/A289R/T065S, T059K/F063L/S066N/S129V,T004L/S023W, N050F/T054H, T059R/S066Q/S129V, V190I/D220E/S265W/L282F,T004S/S023Y/G024M, T004L/S023N/G024Y, T059K/S066N/S129I,T059R/S066N/S129I, L282M/Q286R/A289R/P162S, N046Q/N050F/T179N,T059K/Y082C/S129V, T059K/S129I, N050Y/T054K, T059K/S066Q/V102A/S129Q,T059R/S066Q/S129I, T059W/S066N/S129V/S290R, T059R/S129I,T059K/S066Q/S129I, T059K/S066Q/S129V,S265P/L282M/Q286R/A289R/T202S/K203N, T004V/S023N/G024W, S265P/Q286K,S265P/L282F/A289R, D220P/S265W, L055F/T059W/S129V, T059R/S129Q/S191R,N050W/T054K, T004S/S023W/G024M, R047K/N050F/T054H, T059K/S066N/K088E,T059K/S066Q/S129I/V291L, L282M/Q286R/A289R, T059R/S066N/F085S/S129I,L282F/Q286P/A289R, L282F/Q286R/A289R, G099D/S265P/L282F/Q286K/A289R,N046Q/N050F, N050Y/T059W/S066N/S129V, T0091/D220P/S265N,V190F/D220P/S265W, N157Y/T263W/A273H/S285R, T263W/A273H/S285R,T263W/S285W, T004V/S023Y, N046Q/R047K/N050W, N050W/T054L,N200Y/S265P/L282F/Q286P/A289R, T059R/S066Q/P264Q, T004V/G024Y,T004L/G024Y, N050Y/S1911, N050Y/T054L, T004L/S023W/G024Y/N155K,F169I/L282F/Q286R/A289R, L282M/Q286K/A289R, F130L/M138L/E152W/D183N,N046Q/R047K/N050Y/T054H, T004V/G024M, N050Y/T059W/S066Q/S129V,S023N/G024Y, T054H/P162Q, T004S/S023W/G024Y, N050Y/T054H,L282F/Q286R/A289R/F1691, R047K/N050W, V190F/D220P, L282M/F173Y,T004L/S023Y, N050W/A288D, V190I/D220P/S265Q, S265P/L282F/Q286P/A289R,S265P/L282F/Q286R/A289R, N046Q/N050Y/T054K, T059W/S066Q,T263W/A273H/S285W T263W/A273H/S285P, S023Y/G024M, T004L/S023N/G024W,T004V/S023N/G024Y, T059W/S066N/S129Q, T004S/S023Y, T004S/S023N/G024M,T059W/S066N/A070T, T059W/S066Q/S129Q, T263W/A273H, A273H/285P,N046Q/R047K/N050Y/T054L, N046Q/R047K/N050Y, R047K/N050Y, T263H/S285W,R047K/N050F, N046Q/R047K/N050F/T054H, S023N/G024M, T004S/G024Y,R047K/N050Y/T054H, T059W/S066N/S129I, R047K/T054L, T004S/S023W/G024W,M138L/E152F/T146S, D220P/S265N, T004S/G024M, T004V/S023N,N046Q/N050F/T054K, N046Q/N050Y/T054H, Q062H/S066Q/S129Q, T059W/S129Q,T059W/S129V, N050F/T054K, R047K/N050F/T054L, V190I/D220P/S265W,N1121/T263H/A273H/S285R, T059W/S066N/S129V, T059W/S066Q/S129I,T059W/S129I, T263W/S285P, V190I/D220P, A289V/T263H/A273H,T263H/A273H/S285P, N90S/A273H/S285P, R047K/N050Y/T054L, T004S/S023N,T059R/S129Q, N046Q/R047K/T054H, T059W/S066Q/S129V, E152W/T179P,N050Y/S066Q/S129V, T202S/T263W/A273H, T263W/A273H/S285P,M138L/E152W/T179P, N046Q/R047K, N046Q/T054H/F176L, T004L/G024M,T004S/L282M, T263H/A273H, T263H/A273H/S285W, T004L/S023Y/G024M,L282F/Q286P, T004V/S023Y/G024Y, V190F/S265W, M138L/E152F,V190F/D220E/S265W, N046Q/N050F/T054H, N157Y/S285W, T004F/S023Y/G024M,T004V/S023N/G024M, L1981/D220E/S265Q, N046Q/N050Y/T054K/A154T,S016L/D220E/S265W, D220E/S265W, D220E/A237S/S265W, S066Q/S129Q,V190F/D220E/S265Q/T2671, L282M/F173Y/T219S, E152F/T179P, V190I/S265W,M138L/S066Q, M138L/E152W, T059W/S066Q/A070T/S129I, V190F/D220E/S265N,V190F/S265N, N046Q/N050Y, and M138L/E152F/T179P.

In yet further embodiments, the present invention provides isolatedvariant neutral metalloproteases, wherein the metalloprotease comprisesmultiple mutations selected from V190I/D220P, V190I/D220P/S265Q,V190L/D220E, V190I/D220E/S265Q, V190I/D220E/S265W/L282F,V190L/D220E/S265Q, V190I/D220E/S265W, V190L/D220E/S265N,T059R/S066Q/S129I, V190I/D220E/S265N, V190L/D220E/S265W, V190I/D220E,T059W/S066N/S129V, T059K/S066Q/S129V, T059K/Y082C/S129V,T059R/S066N/S129I, S066Q/S129V, T059R/S066Q/S129V, T059R/S129I,N050Y/T059W/S066N/S129V, D220P/S265N, S066Q/S129I, T059W/S066Q/S129V,T059K/S066Q/S129I, T059R/S129V, N050Y/S066Q/S129V, T059W/S066Q/S129I,N050Y/T059W/S066Q/S129V, T059K/S129I, D220P/S265W, F130L/M138L/T 179P,S066N/S129I, T059R/S066N/S129V, F130I/M138L/T179P,T059R/S066Q/N092S/S129I, S066N/S129V, D220E/S265Q,F130L/M138L/E152W/T179P, T059W/S129V, S265P/L282M/Q286R/A289R,S265P/L282F/Q286R/A289R, T059W/S066N/S129I, V190I/D220P/S265W,F130L/E152W/T179P, F130L/M138L/E152F/T179P, Q062K/S066Q/S129I,T059K/S066N/S129I, E152H/T179P, S265P/L282M/Q286K/A289R,F130L/M138L/E152H/T179P, T263W/A273H/S285R, D220E/S265N,F130I/M138L/E152H/T179P, F130V/M138L/E152W/T179P,F130I/M138L/E152W/T179P, T059W/S129I, D220E/S265W, F130V/M138L/T179P,F130L/E152V/T179P, T059R/S129Q, T263W/S285P, F130I/M138L/E152F/T179P,E152W/T179P, V190L/S265Q, F130L/E152F/T179P, L282M/Q286R/A289R/P162S,D220P/S265Q, M138L/E152F/T179P, F130I/L52H/T179P, M138L/E152W/T179P,F130L/T179P, F130L/M138L/E152W/T179P/Q286H, F130L/M138L/E152H,T263W/A273H/S285W, S265P/Q286K, T059W/S066Q/S129Q, T263W/S285R,T059W/S066N/S129Q, T263W/S285W, T059R/S066N/S129Q,S265P/L282M/Q286R/A289R/T202S/K203N, T059W/S129Q, Q062H/S066Q/S129Q,L282M/Q286R/A289R, V190L/D220E/S265N/V291I, V190L/S265N,F130L/M138L/E152W, N050Y/T059R/S129Q, F130I/T179P, T059K/S066Q/S129Q,T059K/S129Q, S265P/L282M/Q286P/A289R, S265P/L282F/Q286P/A289R,T263W/A273H/S285P, S265P/L282M/Q286K, S016L/D220E/S265W, S066Q/S129Q,S265P/L282M/Q286P, L282F/Q286R/A289R, F130V/E152W/T179P,L044Q/T263W/S285R L055F/T059W/S129V, V190L/S265W, Q286R/A289R,G99D/S265P/L282F/Q286K/A289R, F130L/M138L/E152F, T059R/S066Q/S129Q,F130L/E152H, S066N/S129Q, T004S/S023N/G024M/K269N, S265P/L282M,E152F/T179P, T059W/S066N/S129V/S290R, L282F/Q286K/A289R, F130L/M138L,F130I/M138L/E152W, S265P/L282F, F130I/M138L/E152H, F130V/M138L/E152H,V190I/S265Q, M138L/E152M, S265P/L282F/Q286P, M 138L/E152H,T059K/S066N/K088E, V190I/S265W, F130L/E152W, L282M/Q286K/A289R,L282M/Q286K/A289R/1253V, T263W/A273H, V190I/S265N, M138L/E152W,A273H/S285R, F130I/M138L, F130L/E152F, F130V/M138L/E152W,T059K/S066Q/V102A/S129Q, F130V/E152H/T179P, F130I/M138L/E152F,F130V/M138L/E152F, M138L/E152F, L282M/Q286R, F130I/E152H,S265P/L282F/A289R/T065S, T263H/A273H/S285R, F130V/M138L,T014M/T059R/S129V, L282M/Q286R/A289R/K11N, A273H/S285P,L282M/Q286K/A289R/S132T, T263H/A273H/S285W, F130V/E152W,S265P/L282F/Q286K/N061Y, F130I/E152W, L1981/D220E/S265Q, V190I/S265L,T263H/S285W, S265P/L282F/A289R, M138L/S066Q, F130I/E152F,N90S/A273H/S285P, S032T/T263H/A273H/S285R, L282F/Q286P/A289R,N157Y/T263W/A273H/S285R, V105A/S129V, T263H/A273H/S285P, S129Q/L282H,T059W/S066Q, F130V/E152H, S023W/G024Y, T004V/S023N, T059R/S066Q,N050W/T054L, L282M/Q286P/A289R, A115V/V190L/S265W, L282M/Q286K,T059R/S066N, L282F/Q286P, T004V/S023W/G024M, S265P/L282F/Q286R/L78H,L282F/Q286K, T004V/S023W/G024Y, S023W/G024M, T059R/R256S, F130V/E152F,T004V/G024W, N050W/T054K, S023Y/G024M, T004V/S023Y, T004V/S023Y/G024M,N050Y/T054H, S023W/G024W, T004V/S023Y/G024Y, T004V/S023N/G024W,F130L/M138L/E152F/T179P/V291I, N050Y/T059K/S066Q, T004V/S023Y/G024W,T059K/S066N, T004V/S023N/G024Y, S023Y/G024W, N050F/T054L, R047K/T054K,S023N/G024W, L282M/A289R, S023Y/G024Y, T004V/G024M, R047K/N050F/T054K,N050F/T054K, T059K/S066Q, S023N/G024M, S023N/G024Y, T004L/S023N,R047K/N050W/T054H, T004L/S023W/G024Y, T004S/S023W,N046Q/N050W/T054H/A142T, T004L/S023Y, T004V/S023W, N050W/T054H,T004S/S023N, T004S/L282M, T004L/S023W, N050F/T054H, N050Y/T054L, andR047K/N050W/T054K.

In yet further embodiments, the present invention provides isolatedneutral metalloproteases comprising multiple mutations selected fromS066Q/S129V, S066Q/S129I, N050Y/S066Q/S129V, S066N/S129I,T059K/S066Q/S129V, S066N/S129V, F130L/E152W/T179P,S265P/L282M/Q286R/A289R, F130L/E152V/T179P, T059K/S066Q/S129I,T263W/S285P, T059K/S066N/S129I, T263W/A273H/S285P,S265P/L282F/Q286R/A289R, F130V/E152W/T179P, T263W/A273H/S285R,V190I/D220P/S265W, F130L/E152H, S066N/S129Q, S265P/L282M/Q286K/A289R,V190I/D220E, T059R/S066N/S129I, V190I/D220E/S265W, T059K/S129I,T059R/S066Q/S129I, F130I/M138L/E152H/T179P, F130I/T179P,T263W/A273H/S285W, S016L/D220E/S265W, S066Q/S129Q, V190I/D220E/S265Q,T059R/S066Q/S129V, D220E/S265N, V190L/D220E, D220E/S265W, V190I/D220P,V190L/D220E/S265N, L044Q/T263W/S285R, S265P/L282M/Q286P/A289R,F130L/M138L/E152H/T179P, T263W/S285R, L282M/Q286R/A289R, T263W/S285W,F130I/E152H/T179P, V190I/D220E/S265N, V190L/D220E/S265W,V190I/D220P/S265Q, T059R/S066N/S129V, V190L/D220E/S265Q, E152H/T179P,F130L/M138L/E152F/T179P, Q062H/S066Q/S129Q, T059R/S129V,V190I/D220E/S265W/L282F, V190I/S265Q, F130L/E152F/T179P, D220E/S265Q,E152W/T179P, T059K/S066Q/S129Q, F130L/M138L/T179P,F130I/M138L/E152F/T179P, F130L/M138L/E152W/T179P,N050Y/T059W/S066Q/S129V, S265P/L282M/Q286K, T059R/S129I,F130V/E152H/T179P, D220P/S265N, S265P/L282M/Q286P, F130I/E52H,T059R/S066Q/N092S/S129I, F130L/T179P, G99D/S265P/L282F/Q286K/A289R,T263W/A273H, V190I/S265N, D220P/S265W, F130L/E152W, F130L/M138L/E152H,S265P/L282M, V190I/S265Q, F130L/E152F, T059K/S129Q, Q286R/A289R,M138L/E152W/T179P, F130I/M138L/E152H, D220P/S265Q, V190L/S265N,F130I/M138L/E152W, S265P/Q286K, V190L/S265Q, V190I/S265W,F130L/M138L/E152F, F130V/E152H, E152F/T179P, N050Y/T059W/S066N/S129V,T059R/S066N/S129Q, F1301/L152W, F130V/E152W, T059R/S066Q/S129Q,T263H/A273H/S285P, N90S/A273H/S285P, V190L/D220E/S265N/V291I,T059R/S129Q, A273H/S285P, F130I/M138L/E152W/T179P, F130V/M138L/E152F,N050Y/T059R/S129Q, T059W/S066Q/S129I, F130V/M138L/T179P,F130V/M138L/E152W/T179P, V190L/S265W, F130V/M138L/E152W,T059W/S066Q/S129V, V190I/S265Q, F130V/M138L/E152H, F130I/E152F,N157Y/T263W/A273H/S285R, T263H/S285W, M138L/E152F/T179P,A115V/V190L/S265W, M138L/E152M, T263H/A273H/S285W, F130L/M138L/E152W,T059K/S066N/K088E, F130I/M138L/E152F, F130/M138L/T179P, T004V/S023N,T059K/S066Q/V102A/S129Q, F130L/M138L, N047K/N050F/T054K,T263H/A273H/S285R, F130L/M138L/E152W/T179P/Q286H, M138L/E152H,M138L/S066Q, L282M/Q286R/A289R/P162S, L282F/Q286R/A289R,Q062K/S066Q/S129I, A273H/S285R, S265P/L282F/Q286P,S265P/L282F/Q286P/A289R, S265P/L282M/Q286R/A289R/T202S/K203N,T059W/S066N/S129I, V190/S265L, T059W/S066N/S129V, F130I/M138L,L282M/Q286K/A289R/1253V, R047K/N050F/T054K, M138L/E152F, N050W/T054K,L1981/D220E/S265Q, L282F/Q286K/A289R, N050F/T054K, L282M/Q286R,M138L/E152W, S265P/L282F, F130V/E152F, T059W/S066N/S129Q, F130V/M138L,T263H/A273H, L282M/Q286K/A289R, N046Q/N050W/T054H/A142T,T059W/S066Q/S129Q, S265P/L282F/A289R/T065S, N050F/T054H, S129Q/L282H,L282M/Q286K/A289R/S132T, L282M/Q286R/A289R/K11N, T059K/S066N,R047K/N050W/T054K, T059K/S066Q, T004V/S023Y, T059W/S066N/S129V/S290R,N050Y/T059K/S066Q, and R047K/N050Y.

The present invention also provides isolated polynucleotides comprisinga nucleotide sequence (i) having at least 70% identity to SEQ ID NOS:1,2, 12 and/or 13, or (ii) being capable of hybridizing to a probe derivedfrom any of the nucleotide sequence set forth herein, including theprimer sequences provided in the Examples, under conditions ofintermediate to high stringency, or (iii) being complementary to thenucleotide sequence set forth in SEQ ID NOS:1, 2, 12, and/or 13. In someembodiments, the present invention provides expression vectors encodingat least one such polynucleotide. In further embodiments, the presentinvention provides host cells comprising these expression vectors. Insome particularly preferred embodiments, the host cells are Bacillus sp.The present invention also provides the neutral metalloproteasesproduced by the host cells. In further embodiments, the presentinvention provides polynucleotides that are complementary to at least aportion of the sequence set forth in SEQ ID NOS:1, 2, 12, and/or 13.

The present invention also provides methods of producing an enzymehaving neutral metalloprotease activity, comprising: transforming a hostcell with an expression vector comprising a polynucleotide having atleast 70% sequence identity to SEQ ID NO:1, 2, 12 and/or 13; cultivatingthe transformed host cell under conditions suitable for the host cell.In some preferred embodiments, the host cell is a Bacillus species.

The present invention also provides probes comprising 4 to 150nucleotide sequence substantially identical to a corresponding fragmentof SEQ ID NOS:1, 2, 12, and/or 13, wherein the probe is used to detect anucleic acid sequence coding for an enzyme having metalloproteolyticactivity. In some embodiments, the nucleic acid sequence is obtainedfrom a Bacillus sp.

The present invention also provides cleaning compositions comprising atleast one neutral metalloprotease obtained from a Bacillus sp. In someembodiments, at least one neutral metalloprotease is obtained from B.amyloliquefaciens. In some particularly preferred embodiments, at leastone neutral metalloprotease comprises the amino acid sequence set forthin SEQ ID NO:3, 4, and/or 18. In some further embodiments, the presentinvention provides isolated neutral metalloproteases comprising at least45% amino acid identity with neutral metalloprotease comprising SEQ IDNO:3, 4 and/or 18. In some embodiments, the isolated neutralmetalloproteases comprise at least 50% identity, preferably at least55%, more preferably at least 60%, yet more preferably at least 65%,even more preferably at least 70%, more preferably at least 75%, stillmore preferably at least 80%, more preferably 85%, yet more preferably90%, even more preferably at least 95%, and most preferably 99% identitywith SEQ ID NO:3, 4, and/or 18.

The present invention further provides cleaning compositions comprisingat least one neutral metalloprotease, wherein at least one of theneutral metalloproteases has immunological cross-reactivity with theneutral metalloprotease obtained from a Bacillus sp. In some preferredembodiments, the neutral metalloproteases have immunologicalcross-reactivity with neutral metalloprotease obtained from B.amyloliquefaciens. In alternative embodiments, the neutralmetalloproteases have immunological cross-reactivity with neutralmetalloprotease comprising the amino acid sequence set forth in SEQ IDNO:3, 4 and/or 18. In still further embodiments, the neutralmetalloproteases have cross-reactivity with fragments (i.e., portions)of a Bacillus sp. neutral metalloprotease and/or the neutralmetalloprotease comprising the amino acid sequence set forth in SEQ IDNO:3, 4, and/or 18. The present invention further provides cleaningcompositions comprising at least one neutral metalloprotease, whereinthe neutral metalloprotease is a variant neutral metalloprotease havingan amino acid sequence comprising at least one substitution of an aminoacid made at a position equivalent to a position in a Bacillus sp.neutral metalloprotease having an amino acid sequence set forth in SEQID NO:3, 4 and/or 18, particularly B. amyloliquefaciens neutralmetalloprotease.

In yet additional embodiments, the cleaning compositions contain atleast one neutral metalloprotease comprising a set of mutations in SEQID NO:3, 4 and/or 18. In some particularly preferred embodiments, thevariant neutral metalloproteases comprise at least one substitutioncorresponding to the amino acid positions in SEQ ID NO:3, 4, and/or 18,and wherein the variant neutral metalloproteases have better performancein at least one property, as compared to wild-type B. amyloliquefaciensneutral metalloprotease.

The present invention also provides cleaning compositions comprising acleaning effective amount of a metalloproteolytic enzyme, the enzymecomprising an amino acid sequence having at least 70% sequence identityto SEQ ID NO:3, 4, and/or 18, and a suitable cleaning formulation. Insome preferred embodiments, the cleaning compositions further compriseone or more additional enzymes or enzyme derivatives selected from thegroup consisting of proteases, amylases, lipases, mannanases,pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides compositions comprising at least oneneutral metalloprotease obtained from a Bacillus sp., in particular B.amyloliquefaciens, wherein the compositions further comprise at leastone stabilizer. In some embodiments, the stabilizer is selected fromborax, glycerol, zinc ions, calcium ions, and calcium formate. In someembodiments, the present invention provides competitive inhibitorssuitable to stabilize the enzyme of the present invention to anionicsurfactants. In some embodiments, at least one neutral metalloproteaseis obtained from a Bacillus sp. In some particularly preferredembodiments, at least one neutral metalloprotease is obtained from B.amyloliquefaciens. In some particularly preferred embodiments, at leastone neutral metalloprotease comprises the amino acid sequence set forthin SEQ ID NO:3, 4, and/or 18.

The present invention further provides compositions comprising at leastone neutral metalloprotease obtained from a Bacillus sp., wherein theneutral metalloprotease is an autolytically stable variant. In someembodiments, at least one variant neutral metalloprotease is obtainedfrom B. amyloliquefaciens. In some particularly preferred embodiments,at least one variant neutral metalloprotease comprises the amino acidsequence set forth in SEQ ID NO:3, 4, and/or 18.

The present invention also provides cleaning compositions comprising atleast 0.0001 weight percent of the neutral metalloprotease of thepresent invention, and optionally, an adjunct ingredient. In someembodiments, the composition comprises an adjunct ingredient. In somepreferred embodiments, the composition comprises a sufficient amount ofa pH modifier to provide the composition with a neat pH of from about 3to about 5, the composition being essentially free of materials thathydrolyze at a pH of from about 3 to about 5. In some particularlypreferred embodiments, the materials that hydrolyze comprise asurfactant material. In additional embodiments, the cleaning compositionis a liquid composition, while in other embodiments, the cleaningcomposition is a solid composition and in still further embodiments, thecleaning composition is a gel. Indeed, it is not intended that thepresent invention be limited to any particular formulation and/orcomposition, as various formulations and/or compositions find use in thepresent invention. In further embodiments, the surfactant materialcomprises a sodium alkyl sulfate surfactant that comprises an ethyleneoxide moiety.

The present invention additionally provides cleaning compositions thatin addition to at least one neutral metalloprotease of the presentinvention, further comprise at least one acid stable enzyme, thecleaning composition comprising a sufficient amount of a pH modifier toprovide the composition with a neat pH of from about 3 to about 5, thecomposition being essentially free of materials that hydrolyze at a pHof from about 3 to about 5. In further embodiments, the materials thathydrolyze comprise a surfactant material. In some preferred embodiments,the cleaning composition being a liquid composition. In yet additionalembodiments, the surfactant material comprises a sodium alkyl sulfatesurfactant that comprises an ethylene oxide moiety. In some embodiments,the cleaning composition comprises a suitable adjunct ingredient. Insome additional embodiments, the composition comprises a suitableadjunct ingredient. In some preferred embodiments, the compositioncomprises from about 0.001 to about 0.5 weight % of neutralmetalloprotease.

In some alternatively preferred embodiments, the composition comprisesfrom about 0.01 to about 0.1 weight percent of neutral metalloprotease.

The present invention also provides methods of cleaning, the comprisingthe steps of: a) contacting a surface and/or an article comprising afabric with the cleaning composition comprising the neutralmetalloprotease of the present invention at an appropriateconcentration; and b) optionally washing and/or rinsing the surface ormaterial. In alternative embodiments, any suitable composition providedherein finds use in these methods. In some embodiments, the fabriccomprises at least one grass stain. In some particularly preferredembodiments, the cleaning compositions of the present invention find usein removing grass and other stains from fabrics.

The present invention also provides animal feed comprising at least oneneutral metalloprotease obtained from a Bacillus sp. In someembodiments, at least one neutral metalloprotease is obtained from B.amyloliquefaciens. In some particularly preferred embodiments, at leastone neutral metalloprotease comprises the amino acid sequence set forthin SEQ ID NO:18.

The present invention provides an isolated polypeptide havingmetalloproteolytic activity, (e.g., a neutral metalloprotease) havingthe amino acid sequence set forth in SEQ ID NO:18. In some embodiments,the present invention provides isolated polypeptides havingapproximately 40% to 98% identity with the sequence set forth in SEQ IDNO:18. In some preferred embodiments, the polypeptides haveapproximately 50% to 95% identity with the sequence set forth in SEQ IDNO:18. In some additional preferred embodiments, the polypeptides haveapproximately 60% to 90% identity with the sequence set forth in SEQ IDNO:3. In yet additional embodiments, the polypeptides have approximately65% to 85% identity with the sequence set forth in SEQ ID NO:3, 4,and/or 18. In some particularly preferred embodiments, the polypeptideshave approximately 90% to 95% identity with the sequence set forth inSEQ ID NO:3, 4, and/or 18.

The present invention further provides isolated polynucleotides thatencode neutral metalloproteases comprise an amino acid sequencecomprising at least 40% amino acid sequence identity to SEQ ID NO:3, 4,and/or 18. In some embodiments, the neutral metalloproteases have atleast 50% amino acid sequence identity to SEQ ID NO:3, 4 and/or 18. Insome embodiments, the neutral metalloproteases have at least 60% aminoacid sequence identity to SEQ ID NO:3, 4 and/or 18. In some embodiments,the neutral metalloproteases have at least 70% amino acid sequenceidentity to SEQ ID NO:3, 4, and/or 18. In some embodiments, the neutralmetalloproteases have at least 80% amino acid sequence identity to SEQID NO:3, 4, and/or 18. In some embodiments, the neutral metalloproteaseshave at least 90% amino acid sequence identity to SEQ ID NO:3, 4, and/or18. In some embodiments, the neutral metalloproteases have at least 95%amino acid sequence identity to SEQ ID NO:3, 4, and/or 18. The presentinvention also provides expression vectors comprising any of thepolynucleotides provided above.

The present invention further provides host cells transformed with theexpression vectors of the present invention, such that at least oneneutral metalloprotease is expressed by the host cells. In someembodiments, the host cells are bacteria, while in other embodiments,the host cells are fungi.

The present invention also provides isolated polynucleotides comprisinga nucleotide sequence (i) having at least 70% identity to SEQ ID NO:1,2, 12 and/or 13, or (ii) being capable of hybridizing to a probe derivedfrom the nucleotide sequence of SEQ ID NO:1, 2, 12, and/or 13, underconditions of medium to high stringency, or (iii) being complementary tothe nucleotide sequence of SEQ ID NO:1, 2, 12, and/or 13. In someembodiments, the present invention provides vectors comprising suchpolynucleotide. In further embodiments, the present invention provideshost cells transformed with such vectors.

The present invention further provides methods for producing at leastone enzyme having neutral metalloprotease activity, comprising: thesteps of transforming a host cell with an expression vector comprising apolynucleotide comprising at least 70% sequence identity to SEQ ID NO:1,2, 12, and/or 13, cultivating the transformed host cell under conditionssuitable for the host cell to produce the neutral metalloprotease; andrecovering the neutral metalloprotease. In some preferred embodiments,the host cell is a Bacillus sp, while in some alternative embodiments,the host cell is B. amyloliquefaciens.

The present invention also provides fragments (i.e., portions) of theDNA encoding the neutral metalloproteases provided herein. Thesefragments find use in obtaining partial length DNA fragments capable ofbeing used to isolate or identify polynucleotides encoding matureneutral metalloprotease enzyme described herein from B.amyloliquefaciens, or a segment thereof having proteolytic activity. Insome embodiments, portions of the DNA provided in SEQ ID NO:2 find usein obtaining homologous fragments of DNA from other species which encodea neutral metalloprotease or portion thereof having metalloproteolyticactivity.

The present invention further provides at least one probe comprising apolynucleotide substantially identical to a fragment of SEQ ID NOS:1, 2,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or any primer sequence set forthherein, wherein the probe is used to detect a nucleic acid sequencecoding for an enzyme having metalloproteolytic activity, and wherein thenucleic acid sequence is obtained from a bacterial source. In someembodiments, the bacterial source is a Bacillus sp. In some preferredembodiments, the bacterial source is B. amyloliquefaciens.

The present invention further provides compositions comprising at leastone of the neutral metalloproteases provided herein. In some preferredembodiments, the compositions are cleaning compositions. In someembodiments, the present invention provides cleaning compositionscomprising a cleaning effective amount of at least one neutralmetalloprotease comprising an amino acid sequence having at least 40%sequence identity to SEQ ID NO:18 at least 90% sequence identity to SEQID NO:18, and/or having an amino acid sequence of SEQ ID NO:18. In someembodiments, the cleaning compositions further comprise at least onesuitable cleaning adjunct. In some embodiments, the neutralmetalloprotease is derived from a Bacillus sp. In some preferredembodiments, the Bacillus sp., is B. amyloliquefaciens.

In still further embodiments, the cleaning composition further comprisesat least one additional enzymes or enzyme derivatives selected from thegroup consisting of proteases, amylases, lipases, mannanases, andcellulases.

The present invention also provides isolated naturally occurring neutralmetalloproteases comprising an amino acid sequence having at least 45%sequence identity to SEQ ID NO:18, at least 60% sequence identity to SEQID NO:18, at least 75% sequence identity to SEQ ID NO:18, at least 90%sequence identity to SEQ ID NO:18, at least 95% sequence identity to SEQID NO:18, and/or having the sequence identity of SEQ ID NO:18, theneutral metalloprotease being isolated from a Bacillus sp. In someembodiments, the neutral metalloprotease is isolated from B.amyloliquefaciens.

In additional embodiments, the present invention provides engineeredvariants of the neutral metalloproteases of the present invention. Insome embodiments, the engineered variants are genetically modified usingrecombinant DNA technologies, while in other embodiments, the variantsare naturally occurring. The present invention further encompassesengineered variants of homologous enzymes, as well as isolated enzymehomologs. In some embodiments, the engineered variant homologous neutralmetalloproteases are genetically modified using recombinant DNAtechnologies, while in other embodiments, the variant homologous neutralmetalloproteases are naturally occurring.

The present invention also provides methods for producing neutralmetalloproteases, comprising: (a) transforming a host cell with anexpression vector comprising a polynucleotide having at least 70%sequence identity to SEQ ID NO:2, at least 95% sequence identity to SEQID NO:2, and/or having a polynucleotide sequence of SEQ ID NO:2; (b)cultivating the transformed host cell under conditions suitable for thehost cell to produce the neutral metalloprotease; and (c) recovering theneutral metalloprotease. In some embodiments, the host cell is aBacillus species (e.g., B. subtilis, B. clausii, or B. licheniformis).In alternative embodiments, the host cell is a B. amyloliquefaciens

In further embodiments, the present invention provides means to producehost cells that are capable of producing the neutral metalloproteases ofthe present invention in relatively large quantities. In particularlypreferred embodiments, the present invention provides means to produceneutral metalloprotease with various commercial applications wheredegradation or synthesis of polypeptides are desired, including cleaningcompositions, as well as food and/or feed components, textileprocessing, leather finishing, grain processing, meat processing,cleaning, preparation of protein hydrolysates, digestive aids,microbicidal compositions, bacteriostatic compositions, fungistaticcompositions, personal care products (e.g., oral care, hair care, and/orskin care).

The present invention also provides variant neutral metalloproteaseshaving improved performance as compared to wild-type B.amyloliquefaciens neutral metalloprotease. In some preferredembodiments, the improved performance comprises improvedthermostability, as compared to wild-type B. amyloliquefaciens neutralmetalloprotease. In alternative preferred embodiments, the improvedperformance comprises improved performance under lower or higher pHconditions, as compared to wild-type B. amyloliquefaciens neutralmetalloprotease. In additional preferred embodiments, the improvedperformance comprises improved autolytic stability, as compared towild-type B. amyloliquefaciens neutral metalloprotease. In someparticularly preferred embodiments, the enzyme compositions of thepresent invention have comparable or improved wash performance, ascompared to presently used neutral metalloproteases. Other objects andadvantages of the present invention are apparent herein.

DESCRIPTION OF THE FIGURES

FIG. 1 provides a graph showing the results from the determination ofthe affinity constants of purified MULTIFECT® neutral binding proteinfor zinc and calcium cations using the fluorescent dyes Fluo-Zn3 andFluo-3, respectively.

FIG. 2 provides a graph showing inhibition of protease activity of 0.36mg/ml formulated recombinant B. amyloliquefaciens nprE by LinearAlkylbenzene Sulfonate (LAS) assayed using the QuantiCleave™ proteaseassay.

FIG. 3 provides a sequence alignment of various metalloproteasehomologues (SEQ ID NOS:173-181) that find use in the present invention.

FIG. 4 provides a sequence alignment of various metalloproteasehomologues (SEQ ID NOS:182-191) that find use in the present invention.In this Figure, the numbering is for thermolysin (B.thermoproteolyticus). As in FIG. 3, the “*” indicates conservedresidues, “:” indicates conservatively replaced residues, and “.”indicates similar residues.

FIG. 5 provides a sequence alignment of various metalloproteasehomologues (SEQ ID NOS:192-195) identified through homology modeling.

FIG. 6 provides a map of plasmid pJ4:GO1905.

FIG. 7 provides a map of plasmid pJ4: G01906.

FIG. 8 provides a map of plasmid pJ4:G01907.

FIG. 9 provides a map of plasmid pJ4:G01908.

FIG. 10 provides a map of plasmid pJ4:G01909.

FIG. 11 provides a map of plasmid pJ4:G01938.

FIG. 12 provides a map of plasmid pJHT.

FIG. 13 provides a map of plasmid pAC.

FIG. 14 provides a map of pUBnprE.

FIG. 15 provides a schematic showing the amplification of the aprEpromoter and B. subtilis nprE gene fragments.

FIG. 16 provides a map of plasmid pEL501.

FIG. 17 provides a schematic showing the amplification of the aprEpromoter and B. subtilis nprB gene fragments.

FIG. 18 provides a map of plasmid pEL508.

FIG. 19 provides a schematic showing the amplification of the aprEpromoter and B. stearothermophilus nprT gene fragments, used in theproduction of strain EL560.

FIG. 20 provides a diagram showing the construction of strain EL560.

FIG. 21 provides a schematic showing the amplification of the aprEpromoter and B. caldolyticus npr gene fragments, used in the productionof strain EL561.

FIG. 22 provides a diagram showing the construction of strain EL561.

FIG. 23 provides a schematic showing the amplification of the aprEpromoter and B. thuringiensis nprB gene fragments.

FIG. 24 provides a map of plasmid pEL568.

FIG. 25 provides a graph showing results from experiments designed todetermine the long-term storage of 0.36 mg/ml UF concentrate of neutralmetalloprotease (nprE) in TIDE® 2005 base in the presence of zinc andcalcium ions at 32° C. For comparative purposes, results obtained fortesting without salt and excess calcium are provided.

FIG. 26 provides wash performance test data using Terg-O-Tometer (TOM)and varying soiled substrates. Panel A provides results showing thedelta soil removal (%) of subtilisin (BPN′ Y217L) and purifiedMULTIFECT® Neutral on EMPA 116 (fixed and unfixed on cotton) afterwashing at 15° C. in TIDE®-2005 detergent liquid. Panel B providesresults showing the delta soil removal (%) of subtilisin (BPN′ Y217L)and purified MULTIFECT® Neutral on Equest® grass medium soiled on cottonafter washing at 15° C. in TIDE®-2005 detergent liquid. Panel C providesresults showing the delta soil removal (%) of subtilisin (BPN′ Y217L)and purified MULTIFECT® Neutral on CFT C-10 (pigment, oil, milk oncotton) after washing at 15° C. in TIDE®-2005 detergent liquid.

FIG. 27 provides a graph showing the results of DSC scans for 440 ppmNprE and variants obtained using the VP-Cap DSC (MicroCal™).

FIG. 28 provides a graph showing the results for DSC scans for 440 ppmNprE and variants in the presence of 130 mM citrate were obtained usingthe VP-Cap DSC (MicroCal™).

FIG. 29 provides a graph showing the thermal melting points for 440 ppmNprE in the presence of various additives and obtained using the VP-CapDSC (MicroCal™). In this Figure, the horizontal line represents the Tmfor wild-type NprE with no additives.

FIG. 30 provides a graph showing the remaining activity of nprE and nprEhomologs in 25% TIDE® at 25° C., after 90 minutes.

FIG. 31 provides a graph showing the BMI wash performance of nprE andnprE homologs.

FIG. 32 provides a graph showing the results of NprE stabilitymeasurements in various formulation mixes.

FIG. 33 provides graphs (Panels A, B and C showing the rate of NprEinactivation with different % DTPA concentrations at a fixed calciumformate concentration.

FIG. 34 provides graphs (Panels A, B and C) showing the DOE analysissoftware generated prediction profiles of a DTPA and calcium formatecomposition based on response goal (decay rate).

FIG. 35 provides the amino acid sequences (SEQ ID NOS:222-226) for thecitrate-induced autolytic fragments of NprE highlighting the autolysissites. Fragment 1 and 2 are the first clip, Fragment 3-5 represent thesecond clip. The italicized letters represent the sequenced N-terminiand bold letters highlight the peptides that were identified from thein-gel digestion of the respective fragments.

DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions comprising atleast one neutral metalloprotease enzyme that has improved storagestability. In some embodiments, the neutral metalloprotease finds use incleaning and other applications. In some particularly preferredembodiments, the present invention provides methods and compositionscomprising neutral metalloprotease(s) obtained from Bacillus sp. In somemore particularly preferred embodiments, the neutral metalloprotease isobtained from B. amyloliquefaciens. In still further preferredembodiments, the neutral metalloprotease is a variant of the B.amyloliquefaciens neutral metalloprotease. In yet additionalembodiments, the neutral metalloprotease is a homolog of the B.amyloliquefaciens neutral metalloprotease. The present invention findsparticular use in applications including, but not limited to cleaning,bleaching and disinfecting.

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, protein purification, protein engineering, protein and DNAsequencing, and recombinant DNA fields, which are within the skill ofthe art. Such techniques are known to those of skill in the art and aredescribed in numerous texts and reference works (See e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual”, Second Edition (ColdSpring Harbor), [1989]); and Ausubel et al., “Current Protocols inMolecular Biology” [1987]). All patents, patent applications, articlesand publications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For example,Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham,The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991)provide those of skill in the art with a general dictionaries of many ofthe terms used herein. Although any methods and materials similar orequivalent to those described herein find use in the practice of thepresent invention, the preferred methods and materials are describedherein. Accordingly, the terms defined immediately below are more fullydescribed by reference to the Specification as a whole. Also, as usedherein, the singular terms “a,” “an,” and “the” include the pluralreference unless the context clearly indicates otherwise. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context theyare used by those of skill in the art.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

All documents cited are, in relevant part, incorporated herein byreference; the citation of any document is not to be construed as anadmission that it is prior art with respect to the present invention.

As used herein, the term “bleaching” refers to the treatment of amaterial (e.g., fabric, laundry, pulp, etc.) or surface for a sufficientlength of time and under appropriate pH and temperature conditions toeffect a brightening (i.e., whitening) and/or cleaning of the material.Examples of chemicals suitable for bleaching include but are not limitedto ClO₂, H₂O₂, peracids, NO₂, etc.

As used herein, the term “disinfecting” refers to the removal ofcontaminants from the surfaces, as well as the inhibition or killing ofmicrobes on the surfaces of items. It is not intended that the presentinvention be limited to any particular surface, item, or contaminant(s)or microbes to be removed.

As used herein, the term “multimer” refers to two or more proteins orpeptides that are covalently or non-covalently associated and exist as acomplex in solution. A “dimer” is a multimer that contains two proteinsor peptides; a “trimer” contains three proteins or peptides, etc. Asused herein, “octamer” refers to a multimer of eight proteins orpeptides.

As used herein, “personal care products” means products used in thecleaning, bleaching and/or disinfecting of hair, skin, scalp, and teeth,including, but not limited to shampoos, body lotions, shower gels,topical moisturizers, toothpaste, and/or other topical cleansers. Insome particularly preferred embodiments, these products are utilized onhumans, while in other embodiments, these products find use withnon-human animals (e.g., in veterinary applications).

As used herein, “cleaning compositions” and “cleaning formulations,”unless otherwise indicated, refer to compositions that find use in theremoval of undesired compounds from items to be cleaned, such as fabric,dishes, contact lenses, other solid substrates, hair (shampoos), skin(soaps and creams), teeth (mouthwashes, toothpastes) etc. The termencompasses any materials/compounds selected for the particular type ofcleaning composition desired and the form of the product (e.g., liquid,gel, granule, or spray composition), as long as the composition iscompatible with the neutral metalloprotease and other enzyme(s) used inthe composition. The specific selection of cleaning compositionmaterials are readily made by considering the surface, item or fabric tobe cleaned, and the desired form of the composition for the cleaningconditions during use.

The terms further refer to any composition that is suited for cleaning,bleaching, disinfecting, and/or sterilizing any object and/or surface.It is intended that the terms include, but are not limited to detergentcompositions (e.g., liquid and/or solid laundry detergents and finefabric detergents; hard surface cleaning formulations, such as forglass, wood, ceramic and metal counter tops and windows; carpetcleaners; oven cleaners; fabric fresheners; fabric softeners; andtextile and laundry pre-spotters, as well as dish detergents).

Indeed, the term “cleaning composition” as used herein, includes unlessotherwise indicated, granular or powder-form all-purpose or heavy-dutywashing agents, especially cleaning detergents; liquid, gel orpaste-form all-purpose washing agents, especially the so-calledheavy-duty liquid (HDL) types; liquid fine-fabric detergents; handdishwashing agents or light duty dishwashing agents, especially those ofthe high-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car or carpet shampoos, bathroom cleaners; hair shampoos andhair-rinses; shower gels and foam baths and metal cleaners; as well ascleaning auxiliaries such as bleach additives and “stain-stick” orpre-treat types.

As used herein, the terms “detergent composition” and “detergentformulation” are used in reference to mixtures which are intended foruse in a wash medium for the cleaning of soiled objects. In somepreferred embodiments, the term is used in reference to launderingfabrics and/or garments (e.g., “laundry detergents”). In alternativeembodiments, the term refers to other detergents, such as those used toclean dishes, cutlery, etc. (e.g., “dishwashing detergents”). It is notintended that the present invention be limited to any particulardetergent formulation or composition. Indeed, it is intended that inaddition to neutral metalloprotease, the term encompasses detergentsthat contain surfactants, transferase(s), hydrolytic enzymes, oxidoreductases, builders, bleaching agents, bleach activators, bluing agentsand fluorescent dyes, caking inhibitors, masking agents, enzymeactivators, antioxidants, and solubilizers.

As used herein, “Applicant Enzyme” refers to the neutralmetalloproteases of the present invention.

As used herein, “enhanced performance” in a detergent is defined asincreasing cleaning of bleach-sensitive stains (e.g., grass, tea, wine,blood, dingy, etc.), as determined by usual evaluation after a standardwash cycle. In particular embodiments, the neutral metalloprotease ofthe present invention provides enhanced performance in the removal ofcolored stains and soils. In further embodiments, the enzyme of thepresent invention provides enhanced performance in the removal and/ordecolorization of stains.

As used herein the term “hard surface cleaning composition,” refers todetergent compositions for cleaning hard surfaces such as floors, walls,tile, bath and kitchen fixtures, and the like. Such compositions areprovided in any form, including but not limited to solids, liquids,emulsions, etc.

As used herein, “dishwashing composition” refers to all forms forcompositions for cleaning dishes, including but not limited to granularand liquid forms.

As used herein, “fabric cleaning composition” refers to all forms ofdetergent compositions for cleaning fabrics, including but not limitedto, granular, liquid and bar forms.

As used herein, “textile” refers to woven fabrics, as well as staplefibers and filaments suitable for conversion to or use as yarns, woven,knit, and non-woven fabrics. The term encompasses yarns made fromnatural, as well as synthetic (e.g., manufactured) fibers.

As used herein, “textile materials” is a general term for fibers, yarnintermediates, yarn, fabrics, and products made from fabrics (e.g.,garments and other articles).

As used herein, “fabric” encompasses any textile material. Thus, it isintended that the term encompass garments, as well as fabrics, yarns,fibers, non-woven materials, natural materials, synthetic materials, andany other textile material.

As used herein, the term “compatible,” means that the cleaningcomposition materials do not reduce the enzymatic activity of theneutral metalloprotease to such an extent that the neutralmetalloprotease is not effective as desired during normal usesituations. Specific cleaning composition materials are exemplified indetail hereinafter.

As used herein, “effective amount of enzyme” refers to the quantity ofenzyme necessary to achieve the enzymatic activity required in thespecific application (e.g., personal care product, cleaning composition,etc.). Such effective amounts are readily ascertained by one of ordinaryskill in the art and are based on many factors, such as the particularenzyme variant used, the cleaning application, the specific compositionof the cleaning composition, and whether a liquid or dry (e.g.,granular, bar) composition is required, and the like.

As used herein, “non-fabric cleaning compositions” encompass hardsurface cleaning compositions, dishwashing compositions, personal carecleaning compositions (e.g., oral cleaning compositions, denturecleaning compositions, personal cleansing compositions, etc.), andcompositions suitable for use in the pulp and paper industry.

As used herein, “oral cleaning compositions” refers to dentifrices,toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouthgels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxispastes, dental treatment solutions, and the like.

As used herein, the term “transferase” refers to an enzyme thatcatalyzes the transfer of functional compounds to a range of substrates.

As used herein, “leaving group” refers to the nucleophile which iscleaved from the acyl donor upon substitution by another nucleophile.

As used herein, the term “enzymatic conversion” refers to themodification of a substrate to an intermediate or the modification of anintermediate to an end-product by contacting the substrate orintermediate with an enzyme. In some embodiments, contact is made bydirectly exposing the substrate or intermediate to the appropriateenzyme. In other embodiments, contacting comprises exposing thesubstrate or intermediate to an organism that expresses and/or excretesthe enzyme, and/or metabolizes the desired substrate and/or intermediateto the desired intermediate and/or end-product, respectively.

As used herein, the phrase “detergent stability” refers to the stabilityof a detergent composition. In some embodiments, the stability isassessed during the use of the detergent, while in other embodiments,the term refers to the stability of a detergent composition duringstorage.

As used herein, the phrase, “stability to proteolysis” refers to theability of a protein (e.g., an enzyme) to withstand proteolysis. It isnot intended that the term be limited to the use of any particularprotease to assess the stability of a protein.

As used herein, “oxidative stability” refers to the ability of a proteinto function under oxidative conditions. In particular, the term refersto the ability of a protein to function in the presence of variousconcentrations of H₂O₂ and/or peracid. Stability under various oxidativeconditions can be measured either by standard procedures known to thosein the art and/or by the methods described herein. A substantial changein oxidative stability is evidenced by at least about a 5% or greaterincrease or decrease (in most embodiments, it is preferably an increase)in the half-life of the enzymatic activity, as compared to the enzymaticactivity present in the absence of oxidative compounds.

As used herein, “pH stability” refers to the ability of a protein tofunction at a particular pH. In general, most enzymes have a finite pHrange at which they will function. In addition to enzymes that functionin mid-range pHs (i.e., around pH 7), there are enzymes that are capableof working under conditions with very high or very low pHs. Stability atvarious pHs can be measured either by standard procedures known to thosein the art and/or by the methods described herein. A substantial changein pH stability is evidenced by at least about 5% or greater increase ordecrease (in most embodiments, it is preferably an increase) in thehalf-life of the enzymatic activity, as compared to the enzymaticactivity at the enzyme's optimum pH. However, it is not intended thatthe present invention be limited to any pH stability level nor pH range.

As used herein, “thermal stability” refers to the ability of a proteinto function at a particular temperature. In general, most enzymes have afinite range of temperatures at which they will function. In addition toenzymes that work in mid-range temperatures (e.g., room temperature),there are enzymes that are capable of working in very high or very lowtemperatures. Thermal stability can be measured either by knownprocedures or by the methods described herein. A substantial change inthermal stability is evidenced by at least about 5% or greater increaseor decrease (in most embodiments, it is preferably an increase) in thehalf-life of the catalytic activity of a mutant when exposed to adifferent temperature (i.e., higher or lower) than optimum temperaturefor enzymatic activity. However, it is not intended that the presentinvention be limited to any temperature stability level nor temperaturerange.

As used herein, the term “chemical stability” refers to the stability ofa protein (e.g., an enzyme) towards chemicals that adversely affect itsactivity. In some embodiments, such chemicals include, but are notlimited to hydrogen peroxide, peracids, anionic detergents, cationicdetergents, non-ionic detergents, chelants, etc. However, it is notintended that the present invention be limited to any particularchemical stability level nor range of chemical stability.

As used herein, the phrase “neutral metalloprotease activityimprovement” refers to the relative improvement of neutralmetalloprotease activity, in comparison with a standard enzyme. In someembodiments, the term refers to an improved rate of product formation,while in other embodiments, the term encompasses compositions thatproduce less hydrolysis product. In additional embodiments, the termrefers to neutral metalloprotease compositions with altered substratespecificity.

As used herein, the phrase “alteration in substrate specificity” refersto changes in the substrate specificity of an enzyme. In someembodiments, a change in substrate specificity is defined as adifference between the K_(cat)/K_(m) ratio observed with an enzymecompared to enzyme variants or other enzyme compositions. Enzymesubstrate specificities vary, depending upon the substrate tested. Thesubstrate specificity of an enzyme is determined by comparing thecatalytic efficiencies it exhibits with different substrates. Thesedeterminations find particular use in assessing the efficiency of mutantenzymes, as it is generally desired to produce variant enzymes thatexhibit greater ratios for particular substrates of interest. However,it is not intended that the present invention be limited to anyparticular substrate composition nor any specific substrate specificity.

As used herein, “surface property” is used in reference to anelectrostatic charge, as well as properties such as the hydrophobicityand/or hydrophilicity exhibited by the surface of a protein.

As used herein, the phrase “is independently selected from the groupconsisting of . . . . ” means that moieties or elements that areselected from the referenced Markush group can be the same, can bedifferent or any mixture of elements as indicated in the followingexample:

A molecule having 3 R groups wherein each R group is independentlyselected from the group consisting of A, B and C. Here the three Rgroups may be: AAA, BBB, CCC, AAB, AAC, BBA, BBC, CCA, CCB, or ABC.

In reference to chemical compositions, the term “substituted” as usedherein, means that the organic composition or radical to which the termis applied is:

-   -   (a) made unsaturated by the elimination of at least one element        or radical; or    -   (b) at least one hydrogen in the compound or radical is replaced        with a moiety containing one or more (i) carbon, (ii)        oxygen, (iii) sulfur, (iv) nitrogen or (v) halogen atoms; or    -   (c) both (a) and (b).        Moieties which may replace hydrogen as described in (b)        immediately above, that contain only carbon and hydrogen atoms,        are hydrocarbon moieties including, but not limited to, alkyl,        alkenyl, alkynyl, alkylidenyl, cycloalkyl, phenyl, alkyl phenyl,        naphthyl, anthryl, phenanthryl, fluoryl, steroid groups, and        combinations of these groups with each other and with polyvalent        hydrocarbon groups such as alkylene, alkylidene and alkylidyne        groups. Moieties containing oxygen atoms that may replace        hydrogen as described in (b) immediately above include, but are        not limited to, hydroxy, acyl or keto, ether, epoxy, carboxy,        and ester containing groups. Moieties containing sulfur atoms        that may replace hydrogen as described in (b) immediately above        include, but are not limited to, the sulfur-containing acids and        acid ester groups, thioether groups, mercapto groups and        thioketo groups. Moieties containing nitrogen atoms that may        replace hydrogen as described in (b) immediately above include,        but are not limited to, amino groups, the nitro group, azo        groups, ammonium groups, amide groups, azido groups, isocyanate        groups, cyano groups and nitrile groups. Moieties containing        halogen atoms that may replace hydrogen as described in (b)        immediately above include chloro, bromo, fluoro, iodo groups and        any of the moieties previously described where a hydrogen or a        pendant alkyl group is substituted by a halo group to form a        stable substituted moiety.

It is understood that any of the above moieties (b)(i) through (b)(v)can be substituted into each other in either a monovalent substitutionor by loss of hydrogen in a polyvalent substitution to form anothermonovalent moiety that can replace hydrogen in the organic compound orradical.

As used herein, the terms “purified” and “isolated” refer to the removalof contaminants from a sample. For example, neutral metalloprotease arepurified by removal of contaminating proteins and other compounds withina solution or preparation that are not neutral metalloprotease. In someembodiments, recombinant neutral metalloprotease are expressed inbacterial or fungal host cells and these recombinant neutralmetalloproteases are purified by the removal of other host cellconstituents; the percent of recombinant neutral metalloproteasepolypeptides is thereby increased in the sample. In particularlypreferred embodiments, the metalloprotease of the present invention issubstantially purified to a level of at least about 99% of the proteincomponent, as determined by SDS-PAGE or other standard methods known inthe art. In alternative preferred embodiments, the metalloprotease ofthe present invention comprise at least about 99% of the proteasecomponent of the compositions. In yet alternative embodiments, themetalloprotease is present in a range of about at least 90-95% of thetotal protein and/or protease.

As used herein, “protein of interest,” refers to a protein (e.g., anenzyme or “enzyme of interest”) which is being analyzed, identifiedand/or modified. Naturally-occurring, as well as recombinant proteinsfind use in the present invention.

As used herein, “protein” refers to any composition comprised of aminoacids and recognized as a protein by those of skill in the art. Theterms “protein,” “peptide” and polypeptide are used interchangeablyherein. Wherein a peptide is a portion of a protein, those skilled inthe art understand the use of the term in context.

As used herein, functionally and/or structurally similar proteins areconsidered to be “related proteins.” In some embodiments, these proteinsare derived from a different genus and/or species, including differencesbetween classes of organisms (e.g., a bacterial protein and a fungalprotein). In some embodiments, these proteins are derived from adifferent genus and/or species, including differences between classes oforganisms (e.g., a bacterial enzyme and a fungal enzyme). In additionalembodiments, related proteins are provided from the same species.Indeed, it is not intended that the present invention be limited torelated proteins from any particular source(s). In addition, the term“related proteins” encompasses tertiary structural homologs and primarysequence homologs (e.g., the neutral metalloprotease of the presentinvention). For example, the present invention encompasses such homologsas those provided in FIGS. 3-5. Additional homologs are contemplated,including but not limited to metalloprotease enzymes obtained from B.cereus, B. cereus E33L, B. caldolyticus, B. pumulis, B. megaterium, Bsubtilis amylosacchariticus, Brevibacillus brevis, Paenibacilluspolymyxa (Bacillus polymyxa), B. stearothermophilus, B. thuringiensis,B. subtilis and S. aureus, as well as aureolysin, extracellularelastase, and neutral protease B. In further embodiments, the termencompasses proteins that are immunologically cross-reactive.

As used herein, the term “derivative” refers to a protein which isderived from a protein by addition of one or more amino acids to eitheror both the C- and N-terminal end(s), substitution of one or more aminoacids at one or a number of different sites in the amino acid sequence,and/or deletion of one or more amino acids at either or both ends of theprotein or at one or more sites in the amino acid sequence, and/orinsertion of one or more amino acids at one or more sites in the aminoacid sequence. The preparation of a protein derivative is preferablyachieved by modifying a DNA sequence which encodes for the nativeprotein, transformation of that DNA sequence into a suitable host, andexpression of the modified DNA sequence to form the derivative protein.

Related (and derivative) proteins comprise “variant proteins.” In somepreferred embodiments, variant proteins differ from a parent protein andone another by a small number of amino acid residues. The number ofdiffering amino acid residues may be one or more, preferably 1, 2, 3, 4,5, 10, 15, 20, 30, 40, 50, or more amino acid residues. In somepreferred embodiments, the number of different amino acids betweenvariants is between 1 and 10. In some particularly preferredembodiments, related proteins and particularly variant proteins compriseat least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% amino acid sequence identity. Additionally, arelated protein or a variant protein as used herein, refers to a proteinthat differs from another related protein or a parent protein in thenumber of prominent regions. For example, in some embodiments, variantproteins have 1, 2, 3, 4, 5, or 10 corresponding prominent regions thatdiffer from the parent protein.

Several methods are known in the art that are suitable for generatingvariants of the enzymes of the present invention, including but notlimited to site-saturation mutagenesis, scanning mutagenesis,insertional mutagenesis, random mutagenesis, site-directed mutagenesis,and directed-evolution, as well as various other recombinatorialapproaches.

Characterization of wild-type and mutant proteins is accomplished viaany means suitable and is preferably based on the assessment ofproperties of interest. For example, pH and/or temperature, as well asdetergent and/or oxidative stability is/are determined in someembodiments of the present invention. Indeed, it is contemplated thatenzymes having various degrees of stability in one or more of thesecharacteristics (pH, temperature, proteolytic stability, detergentstability, and/or oxidative stability) will find use.

As used herein, “expression vector” refers to a DNA construct containinga DNA sequence that is operably linked to a suitable control sequencecapable of effecting the expression of the DNA in a suitable host. Suchcontrol sequences include a promoter to effect transcription, anoptional operator sequence to control such transcription, a sequenceencoding suitable mRNA ribosome binding sites and sequences whichcontrol termination of transcription and translation. The vector may bea plasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, “plasmid,”“expression plasmid,” and “vector” are often used interchangeably as theplasmid is the most commonly used form of vector at present. However,the invention is intended to include such other forms of expressionvectors that serve equivalent functions and which are, or become, knownin the art.

In some preferred embodiments, the neutral metalloprotease gene isligated into an appropriate expression plasmid. The cloned neutralmetalloprotease gene is then used to transform or transfect a host cellin order to express the neutral metalloprotease gene. This plasmid mayreplicate in hosts in the sense that it contains the well-known elementsnecessary for plasmid replication or the plasmid may be designed tointegrate into the host chromosome. The necessary elements are providedfor efficient gene expression (e.g., a promoter operably linked to thegene of interest). In some embodiments, these necessary elements aresupplied as the gene's own homologous promoter if it is recognized,(i.e., transcribed, by the host), a transcription terminator (apolyadenylation region for eukaryotic host cells) which is exogenous oris supplied by the endogenous terminator region of the neutralmetalloprotease gene. In some embodiments, a selection gene such as anantibiotic resistance gene that enables continuous cultural maintenanceof plasmid-infected host cells by growth in antimicrobial-containingmedia is also included.

The following cassette mutagenesis method may be used to facilitate theconstruction of the neutral metalloprotease variants of the presentinvention, although other methods may be used. First, as describedherein, a naturally-occurring gene encoding the neutral metalloproteaseis obtained and sequenced in whole or in part. Then, the sequence isscanned for a point at which it is desired to make a mutation (deletion,insertion or substitution) of one or more amino acids in the encodedneutral metalloprotease. The sequences flanking this point are evaluatedfor the presence of restriction sites for replacing a short segment ofthe gene with an oligonucleotide pool which when expressed will encodevarious mutants. Such restriction sites are preferably unique siteswithin the protein gene so as to facilitate the replacement of the genesegment. However, any convenient restriction site which is not overlyredundant in the neutral metalloprotease gene may be used, provided thegene fragments generated by restriction digestion can be reassembled inproper sequence. If restriction sites are not present at locationswithin a convenient distance from the selected point (from 10 to 15nucleotides), such sites are generated by substituting nucleotides inthe gene in such a fashion that neither the reading frame nor the aminoacids encoded are changed in the final construction. Mutation of thegene in order to change its sequence to conform to the desired sequenceis accomplished by M13 primer extension in accord with generally knownmethods. The task of locating suitable flanking regions and evaluatingthe needed changes to arrive at two convenient restriction sitesequences is made routine by the redundancy of the genetic code, arestriction enzyme map of the gene and the large number of differentrestriction enzymes. Note that if a convenient flanking restriction siteis available, the above method need be used only in connection with theflanking region which does not contain a site.

Once the naturally-occurring DNA and/or synthetic DNA is cloned, therestriction sites flanking the positions to be mutated are digested withthe cognate restriction enzymes and a plurality of endtermini-complementary oligonucleotide cassettes are ligated into thegene. The mutagenesis is simplified by this method because all of theoligonucleotides can be synthesized so as to have the same restrictionsites, and no synthetic linkers are necessary to create the restrictionsites.

As used herein, “corresponding to,” refers to a residue at theenumerated position in a protein or peptide, or a residue that isanalogous, homologous, or equivalent to an enumerated residue in aprotein or peptide.

As used herein, “corresponding region,” generally refers to an analogousposition along related proteins or a parent protein.

The terms “nucleic acid molecule encoding,” “nucleic acid sequenceencoding,” “DNA sequence encoding,” and “DNA encoding” refer to theorder or sequence of deoxyribonucleotides along a strand ofdeoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide (protein)chain. The DNA sequence thus codes for the amino acid sequence.

As used herein, the term “analogous sequence” refers to a sequencewithin a protein that provides similar function, tertiary structure,and/or conserved residues as the protein of interest (i.e., typicallythe original protein of interest). For example, in epitope regions thatcontain an alpha helix or a beta sheet structure, the replacement aminoacids in the analogous sequence preferably maintain the same specificstructure. The term also refers to nucleotide sequences, as well asamino acid sequences. In some embodiments, analogous sequences aredeveloped such that the replacement amino acids result in a variantenzyme showing a similar or improved function. In some preferredembodiments, the tertiary structure and/or conserved residues of theamino acids in the protein of interest are located at or near thesegment or fragment of interest. Thus, where the segment or fragment ofinterest contains, for example, an alpha-helix or a beta-sheetstructure, the replacement amino acids preferably maintain that specificstructure.

As used herein, “homologous protein” refers to a protein (e.g., neutralmetalloprotease) that has similar action and/or structure, as a proteinof interest (e.g., an neutral metalloprotease from another source). Itis not intended that homologs (also referred to herein as “homologues”)be necessarily related evolutionarily. Thus, it is intended that theterm encompass the same or similar enzyme(s) (i.e., in terms ofstructure and function) obtained from different species. In somepreferred embodiments, it is desirable to identify a homolog that has aquaternary, tertiary and/or primary structure similar to the protein ofinterest, as replacement for the segment or fragment in the protein ofinterest with an analogous segment from the homolog will reduce thedisruptiveness of the change.

As used herein, “homologous genes” refers to at least a pair of genesfrom different species, which genes correspond to each other and whichare identical or very similar to each other. The term encompasses genesthat are separated by speciation (i.e., the development of new species)(e.g., orthologous genes), as well as genes that have been separated bygenetic duplication (e.g., paralogous genes). These genes encode“homologous proteins.”

As used herein, “ortholog” and “orthologous genes” refer to genes indifferent species that have evolved from a common ancestral gene (i.e.,a homologous gene) by speciation. Typically, orthologs retain the samefunction during the course of evolution. Identification of orthologsfinds use in the reliable prediction of gene function in newly sequencedgenomes.

As used herein, “paralog” and “paralogous genes” refer to genes that arerelated by duplication within a genome. While orthologs retain the samefunction through the course of evolution, paralogs evolve new functions,even though some functions are often related to the original one.Examples of paralogous genes include, but are not limited to genesencoding trypsin, chymotrypsin, elastase, and thrombin, which are allserine proteinases and occur together within the same species.

As used herein, “wild-type” and “native” proteins are those found innature. The terms “wild-type sequence,” and “wild-type gene” are usedinterchangeably herein, to refer to a sequence that is native ornaturally occurring in a host cell. In some embodiments, the wild-typesequence refers to a sequence of interest that is the starting point ofa protein engineering project. The genes encoding thenaturally-occurring protein may be obtained in accord with the generalmethods known to those skilled in the art. The methods generallycomprise synthesizing labeled probes having putative sequences encodingregions of the protein of interest, preparing genomic libraries fromorganisms expressing the protein, and screening the libraries for thegene of interest by hybridization to the probes. Positively hybridizingclones are then mapped and sequenced.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant oligonucleotide” refers to an oligonucleotidecreated using molecular biological manipulations, including but notlimited to, the ligation of two or more oligonucleotide sequencesgenerated by restriction enzyme digestion of a polynucleotide sequence,the synthesis of oligonucleotides (e.g., the synthesis of primers oroligonucleotides) and the like.

The degree of homology between sequences may be determined using anysuitable method known in the art (See e.g., Smith and Waterman, Adv.Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443[1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988];programs such as GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al., Nucl. Acid Res., 12:387-395 [1984]).

For example, PILEUP is a useful program to determine sequence homologylevels. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle, (Feng and Doolittle, J. Mol. Evol.,35:351-360 [1987]). The method is similar to that described by Higginsand Sharp (Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUPparameters including a default gap weight of 3.00, a default gap lengthweight of 0.10, and weighted end gaps. Another example of a usefulalgorithm is the BLAST algorithm, described by Altschul et al.,(Altschul et al., J. Mol. Biol., 215:403-410, [1990]; and Karlin et al.,Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). One particularly usefulBLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth.Enzymol., 266:460-480 [1996]). parameters “W,” “T,” and “X” determinethe sensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See,Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989])alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

As used herein, “percent (%) nucleic acid sequence identity” is definedas the percentage of nucleotide residues in a candidate sequence that isidentical with the nucleotide residues of the sequence.

As used herein, the term “hybridization” refers to the process by whicha strand of nucleic acid joins with a complementary strand through basepairing, as known in the art.

As used herein, the phrase “hybridization conditions” refers to theconditions under which hybridization reactions are conducted. Theseconditions are typically classified by degree of “stringency” of theconditions under which hybridization is measured. The degree ofstringency can be based, for example, on the melting temperature (Tm) ofthe nucleic acid binding complex or probe. For example, “maximumstringency” typically occurs at about Tm-5° C. (5° below the Tm of theprobe); “high stringency” at about 5-10° below the Tm; “intermediatestringency” at about 10-20° below the Tm of the probe; and “lowstringency” at about 20-25° below the Tm. Alternatively, or in addition,hybridization conditions can be based upon the salt or ionic strengthconditions of hybridization and/or one or more stringency washes. Forexample, 6×SSC=very low stringency; 3×SSC=low to medium stringency;1×SSC=medium stringency; and 0.5×SSC=high stringency. Functionally,maximum stringency conditions may be used to identify nucleic acidsequences having strict identity or near-strict identity with thehybridization probe; while high stringency conditions are used toidentify nucleic acid sequences having about 80% or more sequenceidentity with the probe.

For applications requiring high selectivity, it is typically desirableto use relatively stringent conditions to form the hybrids (e.g.,relatively low salt and/or high temperature conditions are used).

The phrases “substantially similar and “substantially identical” in thecontext of at least two nucleic acids or polypeptides typically meansthat a polynucleotide or polypeptide comprises a sequence that has atleast about 40% identity, more preferable at least about 50% identity,yet more preferably at least about 60% identity, preferably at leastabout 75% identity, more preferably at least about 80% identity, yetmore preferably at least about 90%, still more preferably about 95%,most preferably about 97% identity, sometimes as much as about 98% andabout 99% sequence identity, compared to the reference (i.e., wild-type)sequence. Sequence identity may be determined using known programs suchas BLAST, ALIGN, and CLUSTAL using standard parameters. (See e.g.,Altschul, et al., J. Mol. Biol. 215:403-410 [1990]; Henikoff et al.,Proc. Natl. Acad. Sci. USA 89:10915 [1989]; Karin et al., Proc. Natl.Acad. Sci. USA 90:5873 [1993]; and Higgins et al., Gene 73:237-244[1988]). Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. Also,databases may be searched using FASTA (Pearson et al., Proc. Natl. Acad.Sci. USA 85:2444-2448 [1988]). One indication that two polypeptides aresubstantially identical is that the first polypeptide is immunologicallycross-reactive with the second polypeptide. Typically, polypeptides thatdiffer by conservative amino acid substitutions are immunologicallycross-reactive. Thus, a polypeptide is substantially identical to asecond polypeptide, for example, where the two peptides differ only by aconservative substitution. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

As used herein, “equivalent residues” refers to proteins that shareparticular amino acid residues. For example, equivalent resides may beidentified by determining homology at the level of tertiary structurefor a protein (e.g., neutral metalloprotease) whose tertiary structurehas been determined by x-ray crystallography. Equivalent residues aredefined as those for which the atomic coordinates of two or more of themain chain atoms of a particular amino acid residue of the proteinhaving putative equivalent residues and the protein of interest (N on N,CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nmafter alignment. Alignment is achieved after the best model has beenoriented and positioned to give the maximum overlap of atomiccoordinates of non-hydrogen protein atoms of the proteins analyzed. Thepreferred model is the crystallographic model giving the lowest R factorfor experimental diffraction data at the highest resolution available,determined using methods known to those skilled in the art ofcrystallography and protein characterization/analysis.

As used herein, the terms “hybrid neutral metalloproteases” and “fusionneutral metalloproteases” refer to proteins that are engineered from atleast two different or “parental” proteins. In preferred embodiments,these parental proteins are homologs of one another. For example, insome embodiments, a preferred hybrid neutral metalloprotease or fusionprotein contains the N-terminus of a protein and the C-terminus of ahomolog of the protein. In some preferred embodiment, the two terminalends are combined to correspond to the full-length active protein.

The term “regulatory element” as used herein refers to a genetic elementthat controls some aspect of the expression of nucleic acid sequences.For example, a promoter is a regulatory element which facilitates theinitiation of transcription of an operably linked coding region.Additional regulatory elements include splicing signals, polyadenylationsignals and termination signals.

As used herein, “host cells” are generally prokaryotic or eukaryotichosts which are transformed or transfected with vectors constructedusing recombinant DNA techniques known in the art. Transformed hostcells are capable of either replicating vectors encoding the proteinvariants or expressing the desired protein variant. In the case ofvectors which encode the pre- or prepro-form of the protein variant,such variants, when expressed, are typically secreted from the host cellinto the host cell medium.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means transformation, transduction ortransfection. Means of transformation include protoplast transformation,calcium chloride precipitation, electro oration, naked DNA and the likeas known in the art. (See, Chang and Cohen, Mol. Gen. Genet.,168:111-115 [1979]; Smith et al., Appl. Env. Microbiol., 51:634 [1986];and the review article by Ferrari et al., in Harwood, Bacillus, PlenumPublishing Corporation, pp. 57-72 [1989]).

The term “promoter/enhancer” denotes a segment of DNA which containssequences capable of providing both promoter and enhancer functions (forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions). The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An endogenous enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An exogenous(heterologous) enhancer/promoter is one which is placed in juxtapositionto a gene by means of genetic manipulation (i.e., molecular biologicaltechniques).

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York [1989], pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichhas stably integrated foreign or exogenous DNA into the genomic DNA ofthe transfected cell.

The terms “selectable marker” or “selectable gene product” as usedherein refer to the use of a gene which encodes an enzymatic activitythat confers resistance to an antibiotic or drug upon the cell in whichthe selectable marker is expressed.

As used herein, the terms “amplification” and “gene amplification” referto a process by which specific DNA sequences are disproportionatelyreplicated such that the amplified gene becomes present in a higher copynumber than was initially present in the genome. In some embodiments,selection of cells by growth in the presence of a drug (e.g., aninhibitor of an inhibitable enzyme) results in the amplification ofeither the endogenous gene encoding the gene product required for growthin the presence of the drug or by amplification of exogenous (i.e.,input) sequences encoding this gene product, or both. Selection of cellsby growth in the presence of a drug (e.g., an inhibitor of aninhibitable enzyme) may result in the amplification of either theendogenous gene encoding the gene product required for growth in thepresence of the drug or by amplification of exogenous (i.e., input)sequences encoding this gene product, or both.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “co-amplification” refers to the introductioninto a single cell of an amplifiable marker in conjunction with othergene sequences (i.e., comprising one or more non-selectable genes suchas those contained within an expression vector) and the application ofappropriate selective pressure such that the cell amplifies both theamplifiable marker and the other, non-selectable gene sequences. Theamplifiable marker may be physically linked to the other gene sequencesor alternatively two separate pieces of DNA, one containing theamplifiable marker and the other containing the non-selectable marker,may be introduced into the same cell.

As used herein, the terms “amplifiable marker,” “amplifiable gene,” and“amplification vector” refer to a marker, gene or a vector encoding agene which permits the amplification of that gene under appropriategrowth conditions.

As used herein, the term “amplifiable nucleic acid” refers to nucleicacids which may be amplified by any amplification method. It iscontemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample which is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template which may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

“Template specificity” is achieved in most amplification techniques bythe choice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (See e.g., Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038[1972]). Other nucleic acids are not replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters (See,Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase,the enzyme will not ligate the two oligonucleotides or polynucleotides,where there is a mismatch between the oligonucleotide or polynucleotidesubstrate and the template at the ligation junction (See, Wu andWallace, Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, byvirtue of their ability to function at high temperature, are found todisplay high specificity for the sequences bounded and thus defined bythe primers; the high temperature results in thermodynamic conditionsthat favor primer hybridization with the target sequences and nothybridization with non-target sequences.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the term “target,” when used in reference toamplification methods (e.g., the polymerase chain reaction), refers tothe region of nucleic acid bounded by the primers used for polymerasechain reaction. Thus, the “target” is sought to be sorted out from othernucleic acid sequences. A “segment” is defined as a region of nucleicacid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188,hereby incorporated by reference, which include methods for increasingthe concentration of a segment of a target sequence in a mixture ofgenomic DNA without cloning or purification. This process for amplifyingthe target sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed totheir complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one “cycle”; there canbe numerous “cycles”) to obtain a high concentration of an amplifiedsegment of the desired target sequence. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of the primers with respect to each other, and therefore, thislength is a controllable parameter. By virtue of the repeating aspect ofthe process, the method is referred to as the “polymerase chainreaction” (hereinafter “PCR”). Because the desired amplified segments ofthe target sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified”.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention provides methods and compositions comprising atleast one neutral metalloprotease enzyme that has improved storagestability. In some embodiments, the neutral metalloprotease finds use incleaning and other applications. In some particularly preferredembodiments, the present invention provides methods and compositionscomprising neutral metalloprotease(s) obtained from Bacillus sp. In somemore particularly preferred embodiments, the neutral metalloprotease isobtained from B. amyloliquefaciens. In still further preferredembodiments, the neutral metalloprotease is a variant of the B.amyloliquefaciens neutral metalloprotease. In yet additionalembodiments, the neutral metalloprotease is a homolog of the B.amyloliquefaciens neutral metalloprotease. The present invention findsparticular use in applications including, but not limited to cleaning,bleaching and disinfecting.

Also as described in more detail in the Examples below, the presentinvention provides many advantages for cleaning of a wide range ofobjects, including but not limited to clothing, fabrics, medicaldevices, etc. In addition, the present invention provides compositionsthat are effective in cleaning, bleaching, and disinfecting, over arange of wash temperatures and pHs.

In general, proteases hydrolyze amide linkages of proteins via additionof a water molecule to the peptide bond(s). Cleavage occurs at thecarbonyl-group of the peptide bond. In bacterial species such asBacillus, there are two main classes of extracellular proteases namely,alkaline or serine proteases and neutral metalloproteases.

Neutral metalloendopeptidases (i.e., neutral metalloproteases) (EC3.4.24.4) belong to a protease class that has an absolute requirementfor zinc ions for catalytic activity. These enzymes are optimally activeat neutral pH and are in the 30 to 40 kDa size range. Neutralmetalloproteases bind between two and four calcium ions that contributeto the structural stability of the protein. The bound metal ion at theactive site of metalloproteases is an essential feature that allows theactivation of a water molecule. The water molecule then functions as thenucleophile and cleaves the carbonyl group of the peptide bond.

The neutral zinc-binding metalloprotease family includes the bacterialenzyme thermolysin, and thermolysin-like proteases (“TLPs”), as well ascarboxypeptidase A (a digestive enzyme), and the matrix metalloproteasesthat catalyze the reactions in tissue remodeling and degradation. Theonly well characterized of these proteases, with respect to stabilityand function, is thermolysin and its variants (TLPs). Indeed, muchresearch has been focused on the engineering Bacillus subtilis neutralproteases to increase the thermal stability of the enzyme (See e.g.,Vriend et al., In, Tweel et al. (eds), Stability and Stabilization ofenzymes, Elsevier, pp. 93-99 [1993]).

Most effort has been focused on increasing the stability of the proteaseby altering structural determinants identified through the use ofmolecular modeling suggested to prevent local unfolding processes thatwould result in autolysis of the protein and cause the neutral proteaseto denature at high temperatures (See e.g., van den Burg et al., inHopsu-Havu et al., (eds), Proteolysis in Cell Functions Manipulating theAutolytic Pathway of a Bacillus Protease. Biomedical and Health ResearchVol. 13, IOS Press [1997] p. 576).

Compositions and methods to engineer neutral metalloproteases withimproved characteristics are provided herein. As indicated herein,calcium ions have been reported for other proteases such as thermolysinto prevent autolysis. The B. stearothermophilus neutral protease hasbeen stabilized against autolysis and proteolytic degradation byaddition of calcium (See, Dürrschmidt et al., FEBS J., 272:1523-1534[2005]).

Indeed, the present invention provides compositions and methods suitablefor the engineering of neutral metalloproteases that are independent ofcalcium in order to maintain their structural stability. In someembodiments, engineering prevents the local unfolding in a particularsecondary structural element that may prevent proteolysis.

Natural and engineered proteases, such as subtilisin are often expressedin Bacillus subtilis and several have been applied in detergentformulations to remove proteinaceous stains. Others have been appliedfor example in the baking industry (e.g., thermolysin from Bacillusthermoproteolyticus; See e.g., Galante and Formantici, Curr. OrganicChem., 7, 1399-1422 [2003]). In general, the serine proteases have beenmore widely utilized in detergents, at least partially due to therelative ease with which these proteases can be stabilized.

Indeed, metalloproteases are less frequently used in industry, andparticularly in the detergent industry for a number of reasons. Theseenzymes involve more complex protein systems, as the enzymes have theabsolute requirement for calcium and zinc ions for stability andfunction, respectively. Further, the detergent solution as well as thewater used in the laundry process often contains components that ofteninterfere with the binding of the ions by the enzyme or chelate theseions, resulting in a decrease or loss of proteolytic function anddestabilization of the protease.

In contrast to the currently used metalloprotease enzyme systems, thepresent invention provides neutral metalloproteases that aresufficiently stabilized to facilitate long-term shelf storage in liquidlaundry detergent compositions. In particularly preferred embodiments,the metalloprotease stability and activity are preserved throughcomplexing the enzyme with its obligatory active-site zinc molecule.Importantly, the combination of calcium and zinc ions does not have adeleterious effect on the enzyme's function. In some embodiments, theneutral metalloprotease stabilized is the wild-type metalloprotease fromBacillus amyloliquefaciens (e.g., purified MULTIFECT® Neutral; “PMN”).In alternative preferred embodiments, recombinant neutralmetalloprotease (e.g., Bacillus amyloliquefaciens neutralmetalloprotease cloned into Bacillus subtilis (“nprE”)). In additionalembodiments, metalloproteases with improved stability encompass enzymeswith increased affinity for one or more of the calcium binding sites ofthe enzyme. In preferred embodiments, the neutral metalloproteases ofthe present invention find use in general detergent applications,including but not limited to cold water temperatures, grass stains,and/or low pH conditions.

The present invention provides conditions that stabilize zinc-bindingneutral metalloprotease for increased storage stability in detergentbases and/or compositions. In preferred embodiments, the detergentcompositions comprise at least one metalloprotease (e.g., any Bacillusneutral metalloprotease) that is stabilized against autolysis andunfolding, by the inclusion within the detergent formulation of theessential zinc and/or calcium ions. In some particularly preferredembodiments, the neutral metalloprotease from Bacillus amyloliquefaciens(PMN) and the recombinant form expressed in Bacillus subtilis (nprE)that bind zinc ion with 10-fold greater affinity than the calcium ionfind use in the present invention. The stabilized protease in thepresence of essential zinc ions has improved stability againstproteolysis when compared to the same proteases with in the absence ofions.

Although some experimental results indicated that nprE loses someproteolytic activity (˜20%) after one hour of adding the detergent base,nprE incubated at 32° C. in the presence of zinc ions showed significantstabilization over the test conditions with no additional salts orcalcium ions. The presence of both calcium and zinc ions did not show anadditive effect. At zinc ion concentrations lower than 15 mM neutralmetalloprotease is sufficiently stable over approximately 4 weeks. Thus,the present invention provides compositions comprising the addition ofzinc to increase the storage life of neutral metalloprotease in thepresence of detergent components.

Furthermore, in alternative embodiments, the zinc cation is replacedwith Co²⁺, Mn²⁺ or Fe²⁺, since all of these ions have been shown to bindand restore the protease activity of neutral metalloproteases. However,it was determined that Mn²⁺ and Fe²⁺ do not restore all of the nativeactivity. While Co²⁺ restores the highest percentage of the activity, itis apparently less firmly bound than Zn²⁺. The zinc cation is anessential feature in the active site of all neutral metalloproteases, asit is known to play a role in substrate binding and enzyme catalysis(See e.g., Holmquist and Vallee, J. Biol. Chem., 249:4601-4607 [1974]).The relatively tight affinity of the neutral metalloprotease for thezinc cation (˜μM range) and the approximately 10-fold greater affinityfor this ion relative to calcium, suggest that zinc functions as astabilizer, thereby preventing autolysis, proteolysis and unfolding.However, it is not intended that the present invention be limited to anyparticular mechanisms.

The present invention provides extremely beneficial opportunities forapplication in the production and development of industrial detergents.Many detergents are available with high specificity towards the removalof protein, starch and grease stains. In particular, the better washperformance of PMN or neutral metalloprotease from B. amyloliquefacienson Equest Grass (Warwick) indicates that the neutral metalloproteases ofthe present invention in a detergent base that also contains zinc findsuse in improved detergent compositions.

Detailed Description of Cleaning and Detergent Formulations of thePresent Invention

Unless otherwise noted, all component or composition levels providedherein are made in reference to the active level of that component orcomposition, and are exclusive of impurities, for example, residualsolvents or by-products, which may be present in commercially availablesources.

Enzyme components weights are based on total active protein.

All percentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalcomposition unless otherwise indicated.

In the exemplified detergent compositions, the enzymes levels areexpressed by pure enzyme by weight of the total composition and unlessotherwise specified, the detergent ingredients are expressed by weightof the total compositions.

Cleaning Compositions Comprising Neutral Metalloprotease

The stabilized neutral metalloproteases of the present invention areuseful in formulating various detergent compositions. The cleaningcomposition of the present invention may be advantageously employed forexample, in laundry applications, hard surface cleaning, automaticdishwashing applications, as well as cosmetic applications such asdentures, teeth, hair and skin. However, due to the unique advantages ofincreased effectiveness in lower temperature solutions and the superiorcolor-safety profile, the enzymes of the present invention are ideallysuited for laundry applications such as the bleaching of fabrics.Furthermore, the enzymes of the present invention find use in bothgranular and liquid compositions.

The enzymes of the present invention also find use in cleaning additiveproducts. A cleaning additive product including at least one enzyme ofthe present invention is ideally suited for inclusion in a wash processwhen additional bleaching effectiveness is desired. Such instancesinclude, but are not limited to low temperature solution cleaningapplications. The additive product may be, in its simplest form, one ormore neutral metalloprotease enzyme as provided by the presentinvention. In some embodiments, the additive is packaged in dosage formfor addition to a cleaning process where a source of peroxygen isemployed and increased bleaching effectiveness is desired. In someembodiments, the single dosage form comprises a pill, tablet, gelcap orother single dosage unit including pre-measured powders and/or liquids.In some embodiments, filler and/or carrier material(s) are included, inorder to increase the volume of such composition. Suitable filler orcarrier materials include, but are not limited to, various salts ofsulfate, carbonate and silicate as well as talc, clay and the like. Insome embodiments filler and/or carrier materials for liquid compositionsinclude water and/or low molecular weight primary and secondary alcoholsincluding polyols and diols. Examples of such alcohols include, but arenot limited to, methanol, ethanol, propanol and isopropanol. In someembodiments, the compositions comprise from about 5% to about 90% ofsuch materials. In additional embodiments, acidic fillers are used toreduce the pH of the composition. In some alternative embodiments, thecleaning additive includes at least one activated peroxygen source asdescribed below and/or adjunct ingredients as more fully describedbelow.

The cleaning compositions and cleaning additives of the presentinvention require an effective amount of neutral metalloprotease enzymeas provided in the present invention. In some embodiments, the requiredlevel of enzyme is achieved by the addition of one or more species ofneutral metalloprotease provided by the present invention. Typically,the cleaning compositions of the present invention comprise at least0.0001 weight percent, from about 0.0001 to about 1, from about 0.001 toabout 0.5, or even from about 0.01 to about 0.1 weight percent of atleast one neutral metalloprotease provided by the present invention.

In some preferred embodiments, the cleaning compositions provided hereinare typically formulated such that, during use in aqueous cleaningoperations, the wash water has a pH of from about 5.0 to about 11.5, orin alternative embodiments, even from about 6.0 to about 10.5. In somepreferred embodiments, liquid product formulations are typicallyformulated to have a neat pH from about 3.0 to about 9.0, while in somealternative embodiments the formulation has a neat pH from about 3 toabout 5. In some preferred embodiments, granular laundry products aretypically formulated to have a pH from about 8 to about 11. Techniquesfor controlling pH at recommended usage levels include the use ofbuffers, alkalis, acids, etc., and are well known to those skilled inthe art.

In some particularly preferred embodiments, when at least one neutralmetalloprotease is employed in a granular composition or liquid, theneutral metalloprotease is in the form of an encapsulated particle toprotect the enzyme from other components of the granular compositionduring storage. In addition, encapsulation also provides a means ofcontrolling the availability of the neutral metalloprotease(s) duringthe cleaning process and may enhance performance of the neutralmetalloprotease(s). It is contemplated that the encapsulated neutralmetalloproteases of the present invention will find use in varioussettings. It is also intended that the neutral metalloprotease beencapsulated using any suitable encapsulating material(s) and method(s)known in the art.

In some preferred embodiments, the encapsulating material typicallyencapsulates at least part of the neutral metalloprotease catalyst. Insome embodiments, the encapsulating material is water-soluble and/orwater-dispersible. In some additional embodiments, the encapsulatingmaterial has a glass transition temperature (Tg) of 0° C. or higher (Seee.g., WO 97/11151, particularly from page 6, line 25 to page 7, line 2,for more information regarding glass transition temperatures).

In some embodiments, the encapsulating material is selected from thegroup consisting of carbohydrates, natural or synthetic gums, chitin andchitosan, cellulose and cellulose derivatives, silicates, phosphates,borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes andcombinations thereof. In some embodiments in which the encapsulatingmaterial is a carbohydrate, it is selected from the group consisting ofmonosaccharides, oligosaccharides, polysaccharides, and combinationsthereof. IN some preferred embodiments, the encapsulating material is astarch (See e.g., EP 0 922 499; U.S. Pat. No. 4,977,252. U.S. Pat. No.5,354,559, and U.S. Pat. No. 5,935,826, for descriptions of someexemplary suitable starches).

In additional embodiments, the encapsulating material comprises amicrosphere made from plastic (e.g., thermoplastics, acrylonitrile,methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixturesthereof; commercially available microspheres that find use include, butare not limited to EXPANCEL® [Casco Products, Stockholm, Sweden], PM6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, and Q-CEL® [PQ Corp.,Valley Forge, Pa.], LUXSIL® and SPHERICELI® [Potters Industries, Inc.,Carlstadt, N.J. and Valley Forge, Pa.]).

Processes of Making and Using of Applicants' Cleaning Composition

In some preferred embodiments, the cleaning compositions of the presentinvention are formulated into any suitable form and prepared by anyprocess chosen by the formulator, (See e.g., U.S. Pat. No. 5,879,584,U.S. Pat. No. 5,691,297, U.S. Pat. No. 5,574,005, U.S. Pat. No.5,569,645, U.S. Pat. No. 5,565,422, U.S. Pat. No. 5,516,448, U.S. Pat.No. 5,489,392, and U.S. Pat. No. 5,486,303, for some non-limitingexamples). In some embodiments in which a low pH cleaning composition isdesired, the pH of such composition is adjusted via the addition of anacidic material such as HCl.

Adjunct Materials

While not essential for the purposes of the present invention, in someembodiments, the non-limiting list of adjuncts described herein aresuitable for use in the cleaning compositions of the present invention.Indeed, in some embodiments, adjuncts are incorporated into the cleaningcompositions of the present invention. In some embodiments, adjunctmaterials assist and/or enhance cleaning performance, treat thesubstrate to be cleaned, and/or modify the aesthetics of the cleaningcomposition (e.g., perfumes, colorants, dyes, etc.). It is understoodthat such adjuncts are in addition to the neutral metalloproteases ofthe present invention. The precise nature of these additionalcomponents, and levels of incorporation thereof, depends on the physicalform of the composition and the nature of the cleaning operation forwhich it is to be used. Suitable adjunct materials include, but are notlimited to, surfactants, builders, chelating agents, dye transferinhibiting agents, deposition aids, dispersants, additional enzymes, andenzyme stabilizers, catalytic materials, bleach activators, bleachboosters, hydrogen peroxide, sources of hydrogen peroxide, preformedperacids, polymeric dispersing agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,perfumes, structure elasticizing agents, fabric softeners, carriers,hydrotropes, processing aids and/or pigments. In addition to thoseprovided explicitly herein, additional examples are known in the art(See e.g., U.S. Pat. Nos. 5,576,282, 6,306,812 B1 and 6,326,348 B1). Insome embodiments, the aforementioned adjunct ingredients constitute thebalance of the cleaning compositions of the present invention.

Surfactants—In some embodiments, the cleaning compositions of thepresent invention comprise at least one surfactant or surfactant system,wherein the surfactant is selected from nonionic surfactants, anionicsurfactants, cationic surfactants, ampholytic surfactants, zwitterionicsurfactants, semi-polar nonionic surfactants, and mixtures thereof. Insome low pH cleaning composition embodiments (e.g., compositions havinga neat pH of from about 3 to about 5), the composition typically doesnot contain alkyl ethoxylated sulfate, as it is believed that suchsurfactant may be hydrolyzed by such compositions the acidic contents.

In some embodiments, the surfactant is present at a level of from about0.1% to about 60%, while in alternative embodiments, the level is fromabout 1% to about 50%, while in still further embodiments, the level isfrom about 5% to about 40%, by weight of the cleaning composition.

Builders—In some embodiments, the cleaning compositions of the presentinvention comprise one or more detergent builders or builder systems. Insome embodiments incorporating at least one builder, the cleaningcompositions comprise at least about 1%, from about 3% to about 60% oreven from about 5% to about 40% builder by weight of the cleaningcomposition.

Builders include, but are not limited to, the alkali metal, ammonium andalkanolammonium salts of polyphosphates, alkali metal silicates,alkaline earth and alkali metal carbonates, aluminosilicate builderspolycarboxylate compounds. ether hydroxypolycarboxylates, copolymers ofmaleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, thevarious alkali metal, ammonium and substituted ammonium salts ofpolyacetic acids such as ethylenediamine tetraacetic acid andnitrilotriacetic acid, as well as polycarboxylates such as melliticacid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid,benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, andsoluble salts thereof. Indeed, it is contemplated that any suitablebuilder will find use in various embodiments of the present invention.

Chelating Agents—In some embodiments, the cleaning compositions of thepresent invention contain at least one chelating agent, Suitablechelating agents include, but are not limited to copper, iron and/ormanganese chelating agents and mixtures thereof. In embodiments in whichat least one chelating agent is used, the cleaning compositions of thepresent invention comprise from about 0.1% to about 15% or even fromabout 3.0% to about 10% chelating agent by weight of the subjectcleaning composition.

Deposition Aid—In some embodiments, the cleaning compositions of thepresent invention include at least one deposition aid. Suitabledeposition aids include, but are not limited to polyethylene glycol,polypropylene glycol, polycarboxylate, soil release polymers such aspolytelephthalic acid, clays such as kaolinite, montmorillonite,atapulgite, illite, bentonite, halloysite, and mixtures thereof.

Dye Transfer Inhibiting Agents—In some embodiments, the cleaningcompositions of the present invention include one or more dye transferinhibiting agents. Suitable polymeric dye transfer inhibiting agentsinclude, but are not limited to, polyvinylpyrrolidone polymers,polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone andN-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof.

In embodiments in which at least one dye transfer inhibiting agent isused, the cleaning compositions of the present invention comprise fromabout 0.0001% to about 10%, from about 0.01% to about 5%, or even fromabout 0.1% to about 3% by weight of the cleaning composition.

Dispersants—In some embodiments, the cleaning compositions of thepresent invention contains at least one dispersants. Suitablewater-soluble organic materials include, but are not limited to thehomo- or co-polymeric acids or their salts, in which the polycarboxylicacid comprises at least two carboxyl radicals separated from each otherby not more than two carbon atoms.

Enzymes—In some embodiments, the cleaning compositions of the presentinvention comprise one or more detergent enzymes which provide cleaningperformance and/or fabric care benefits. Examples of suitable enzymesinclude, but are not limited to, hemicellulases, peroxidases, proteases,cellulases, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, keratinases, reductases, oxidases, phenoloxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase,laccase, and amylases, or mixtures thereof. In some embodiments, acombination of enzymes is used (i.e., a “cocktail”) comprisingconventional applicable enzymes like protease, lipase, cutinase and/orcellulase in conjunction with amylase is used.

Enzyme Stabilizers—In some embodiments of the present invention, theenzymes used in the detergent formulations of the present invention arestabilized. It is contemplated that various techniques for enzymestabilization will find use in the present invention. For example, insome embodiments, the enzymes employed herein are stabilized by thepresence of water-soluble sources of zinc (II), calcium (II) and/ormagnesium (II) ions in the finished compositions that provide such ionsto the enzymes, as well as. other metal ions (e.g., barium (II),scandium (II), iron (II), manganese (II), aluminum (III), Tin (II),cobalt (II), copper (II), Nickel (II), and oxovanadium (IV)).

Catalytic Metal Complexes—In some embodiments, the cleaning compositionsof the present invention contain one or more catalytic metal complexes.In some embodiments, a metal-containing bleach catalyst finds use. Insome preferred embodiments, the metal bleach catalyst comprises acatalyst system comprising a transition metal cation of defined bleachcatalytic activity, (e.g., copper, iron, titanium, ruthenium, tungsten,molybdenum, or manganese cations), an auxiliary metal cation havinglittle or no bleach catalytic activity (e.g., zinc or aluminum cations),and a sequestrate having defined stability constants for the catalyticand auxiliary metal cations, particularly ethylenediaminetetraaceticacid, ethylenediaminetetra (methylenephosphonic acid) and water-solublesalts thereof are used (See e.g., U.S. Pat. No. 4,430,243).

In some embodiments, the cleaning compositions of the present inventionare catalyzed by means of a manganese compound. Such compounds andlevels of use are well known in the art (See e.g., U.S. Pat. No.5,576,282).

In additional embodiments, cobalt bleach catalysts find use in thecleaning compositions of the present invention. Various cobalt bleachcatalysts are known in the art (See e.g., U.S. Pat. No. 5,597,936, andU.S. Pat. No. 5,595,967). Such cobalt catalysts are readily prepared byknown procedures (See e.g., U.S. Pat. No. 5,597,936, and U.S. Pat. No.5,595,967).

In additional embodiments, the cleaning compositions of the presentinvention include a transition metal complex of a macropolycyclic rigidligand (“MRL”). As a practical matter, and not by way of limitation, insome embodiments, the compositions and cleaning processes provided bythe present invention are adjusted to provide on the order of at leastone part per hundred million of the active MRL species in the aqueouswashing medium, and in some preferred embodiments, provide from about0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to about10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of theMRL in the wash liquor.

Preferred transition-metals in the instant transition-metal bleachcatalyst include, but are not limited to manganese, iron and chromium.Preferred MRLs also include, but are not limited to special ultra-rigidligands that are cross-bridged (e.g.,5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane). Suitabletransition metal MRLs are readily prepared by known procedures (Seee.g., WO 00/32601, and U.S. Pat. No. 6,225,464).

Processes of Making and Using Cleaning Compositions

The cleaning compositions of the present invention are formulated intoany suitable form and prepared by any suitable process chosen by theformulator, (See e.g., U.S. Pat. No. 5,879,584, U.S. Pat. No. 5,691,297,U.S. Pat. No. 5,574,005, U.S. Pat. No. 5,569,645, U.S. Pat. No.5,565,422, U.S. Pat. No. 5,516,448, U.S. Pat. No. 5,489,392, U.S. Pat.No. 5,486,303, U.S. Pat. No. 4,515,705, U.S. Pat. No. 4,537,706, U.S.Pat. No. 4,515,707, U.S. Pat. No. 4,550,862, U.S. Pat. No. 4,561,998,U.S. Pat. No. 4,597,898, U.S. Pat. No. 4,968,451, U.S. Pat. No.5,565,145, U.S. Pat. No. 5,929,022, U.S. Pat. No. 6,294,514, and U.S.Pat. No. 6,376,445, all of which are incorporated herein by referencefor some non-limiting examples).

Method of Use

In preferred embodiments, the cleaning compositions of the presentinvention find use in cleaning surfaces and/or fabrics. In someembodiments, at least a portion of the surface and/or fabric iscontacted with at least one embodiment of the cleaning compositions ofthe present invention, in neat form or diluted in a wash liquor, andthen the surface and/or fabric is optionally washed and/or rinsed. Forpurposes of the present invention, “washing” includes, but is notlimited to, scrubbing, and mechanical agitation. In some embodiments,the fabric comprises any fabric capable of being laundered in normalconsumer use conditions. In preferred embodiments, the cleaningcompositions of the present invention are used at concentrations of fromabout 500 ppm to about 15,000 ppm in solution. In some embodiments inwhich the wash solvent is water, the water temperature typically rangesfrom about 5° C. to about 90° C. In some preferred embodiments forfabric cleaning, the water to fabric mass ratio is typically from about1:1 to about 30:1.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: ° C. (degrees Centigrade); rpm (revolutions perminute); H₂O (water); HCl (hydrochloric acid); aa and AA (amino acid);bp (base pair); kb (kilobase pair); kD (kilodaltons); gm (grams); μg andug (micrograms); mg (milligrams); ng (nanograms); μl and ul(microliters); ml (milliliters); mm (millimeters); nm (nanometers); μmand um (micrometer); M (molar); mM (millimolar); μM and uM (micromolar);U (units); V (volts); MW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl(sodium chloride); OD₂₈₀ (optical density at 280 nm); OD₄₀₅ (opticaldensity at 405 nm); OD₆₀₀ (optical density at 600 nm); PAGE(polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphatebuffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]);LAS (lauryl sodium sulfonate); SDS (sodium dodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); TAED(N,N,N′N′-tetraacetylethylenediamine); BES (polyesstersulfone); MES(2-morpholinoethanesulfonic acid, monohydrate; f.w. 195.24; Sigma #M-3671); CaCl₂ (calcium chloride, anhydrous; f.w. 110.99; Sigma #C-4901); DMF (N,N-dimethylformamide, f.w. 73.09, d=0.95); Abz-AGLA-Nba(2-Aminobenzoyl-L-alanylglycyl-L-leucyl-L-alamino-4-nitrobenzylamide,f.w. 583.65; Bachem # H-6675, VWR catalog #100040-598); SBG1% (“SuperBroth with Glucose”; 6 g Soytone [Difco], 3 g yeast extract, 6 g NaCl, 6g glucose); the pH was adjusted to 7.1 with NaOH prior to sterilizationusing methods known in the art; w/v (weight to volume); v/v (volume tovolume); Npr and npr (neutral metalloprotease); SEQUEST® (SEQUESTdatabase search program, University of Washington); Npr and npr (neutralmetalloprotease gene); nprE and NprE (B. amyloliquefaciens neutralmetalloprotease); PMN (purified MULTIFECT® metalloprotease); MS (massspectroscopy); SRI (Stain Removal Index); TIGR (The Institute forGenomic Research, Rockville, Md.); AATCC (American Association ofTextile and Coloring Chemists); Amersham (Amersham Life Science, Inc.Arlington Heights, Ill.); Corning (Corning International, Corning,N.Y.); ICN (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.); Pierce(Pierce Biotechnology, Rockford, Ill.); Equest (Equest, WarwickInternational Group, Inc., Flintshire, UK); EMPA (EidgenossischeMaterial Prufungs und Versuch Anstalt, St. Gallen, Switzerland); CFT(Center for Test Materials, Vlaardingen, The Netherlands); Amicon(Amicon, Inc., Beverly, Mass.); ATCC (American Type Culture Collection,Manassas, Va.); Becton Dickinson (Becton Dickinson Labware, LincolnPark, N.J.); Perkin-Elmer (Perkin-Elmer, Wellesley, Mass.); Rainin(Rainin Instrument, LLC, Woburn, Mass.); Eppendorf (Eppendorf AG,Hamburg, Germany); Waters (Waters, Inc., Milford, Mass.); Geneart(Geneart GmbH, Regensburg, Germany); Perseptive Biosystems (PerseptiveBiosystems, Ramsey, Minn.); Molecular Probes (Molecular Probes, Eugene,Oreg.); BioRad (BioRad, Richmond, Calif.); Clontech (CLONTECHLaboratories, Palo Alto, Calif.); Cargill (Cargill, Inc., Minneapolis,Minn.); Difco (Difco Laboratories, Detroit, Mich.); GIBCO BRL or GibcoBRL (Life Technologies, Inc., Gaithersburg, Md.); New Brunswick (NewBrunswick Scientific Company, Inc., Edison, N.J.); Thermoelectron(Thermoelectron Corp., Waltham, Mass.); BMG (BMG Labtech, GmbH,Offenburg, Germany); Greiner (Greiner Bio-One, Kremsmuenster, Austria);Novagen (Novagen, Inc., Madison, Wis.); Novex (Novex, San Diego,Calif.); Finnzymes (Finnzymes OY, Finland) Qiagen (Qiagen, Inc.,Valencia, Calif.); Invitrogen (Invitrogen Corp., Carlsbad, Calif.);Sigma (Sigma Chemical Co., St. Louis, Mo.); DuPont Instruments(Asheville, N.Y.); Global Medical Instrumentation or GMI (Global MedicalInstrumentation; Ramsey, Minn.); MJ Research (MJ Research, Waltham,Mass.); Infors (Infors AG, Bottmingen, Switzerland); Stratagene(Stratagene Cloning Systems, La Jolla, Calif.); Roche (Hoffmann LaRoche, Inc., Nutley, N.J.); Agilent (Agilent Technologies, Palo Alto,Calif.); S-Matrix (S-Matrix Corp., Eureka, Calif.); US Testing (UnitedStates Testing Co., Hoboken, N.Y.); West Coast Analytical Services (WestCoast Analytical Services, Inc., Santa Fe Springs, Calif.); Ion BeamAnalysis Laboratory (Ion Bean Analysis Laboratory, The University ofSurrey Ion Beam Centre (Guildford, UK); TOM (Terg-o-Meter); BMI (blood,milk, ink); BaChem (BaChem AG, Bubendorf, Switzerland); MolecularDevices (Molecular Devices, Inc., Sunnyvale, Calif.); Corning (CorningInternational, Corning, N.Y.); MicroCal (Microcal, Inc., Northhampton,Mass.); Chemical Computing (Chemical Computing Corp., Montreal, Canada);NCBI (National Center for Biotechnology Information); Argo Bioanalytica(Argo Bioanalytica. Inc, New Jersey); Vydac (Grace Vydac, Hesperia,Calif.); Minolta (Konica Minolta, Ramsey, N.J.); and Zeiss (Carl Zeiss,Inc., Thornwood, N.Y.).

In these experiments, a spectrophotometer was used to measure theabsorbance of the products formed after the completion of the reactions.A reflectometer was used to measure the reflectance of the swatches.Unless otherwise indicated, protein concentrations were estimated byCoomassie Plus (Pierce), using BSA as the standard.

The following assays were used in the Examples described below.

A. Bradford Assay for Protein Content Determination in 96-wellMicrotiter Plates (MTPs)

In these assays, the Bradford dye reagent (Quick Start) assay was usedto determine the protein concentration in NprE protease samples on MTPscale.

In this assay system, the chemical and reagent solutions used were:

Quick Start Bradford Dye Reagent BIO-RAD, #500-0205 Dilution buffer 10mM NaCl, 0.1 mM CaCl2, 0.005% TWEEN ®-80

The equipment used was a Biomek FX Robot (Beckman) and a SpectraMAX(type 340) MTP reader; the MTPs were from Costar (type 9017).

In the test, 200 μl Bradford Dye Reagent was pipetted into each well,followed by 15 μl dilution buffer. Finally 10 μl of filtered culturebroth were added to the wells. After thorough mixing, the MTPs wereincubated for at least 10 minutes at room temperature. Possible airbubbles were blown away and the ODs of the wells were read at 595 nm.

To determine the protein concentration, the background reading (i.e.,from uninoculated wells) was subtracted form the sample readings. Theobtained OD₅₉₅ values provide a relative measure of the protein contentin the samples. The linearity of the NprE calibration lines between 0 to5 μg enabled the use of OD₅₉₅ nm values as a relative measure for theprotein content. As the expected content of NprE in supernatant was200-300 μg/ml, the 10 μl sample volume used in the test contains lessthan 5 μg protein, providing values in the linear range.

B. Microswatch Assay for Testing Protease Performance

The detergents used in this assay did not contain enzymes. The equipmentused was a Biomek FX Robot (Beckman) and a SpectraMAX (type 340) MTPreader; the MTPs were from Costar (type 9017).

Detergent Preparation (Cold Water Liquid Detergent; US Conditions):

Milli-Q water was adjusted to 6 gpg water hardness (Ca/Mg=3/1), and 0.78g/l TIDE® 2007-2× detergent was added. The detergent solution wasvigorously stirred for at least 15 minutes. Then. 5 mM HEPES (free acid)was added and the pH adjusted to 8.2.

Microswatches

Microswatches of ¼″ circular diameter were obtained from CFT. Beforecutting of the swatches, the fabric (EMPA 116) was washed with water.Two microswatches were placed in each well of a 96-well microtiter platevertically to expose the whole surface area (i.e., not flat on thebottom of the well).

Test Method

The incubator was set to 20° C. The filtered culture broth samples weretested at an appropriate concentration by dilution with a mixture of 10mM NaCl, 0.1 mM CaCl2 and 0.005% TWEEN®-80 solution. The desireddetergent solution was prepared as described above. Then, 190 μl ofdetergent solution was added to each well of the MTP, containingmicroswatches. To this mixture, 101 of the diluted enzyme solution wereadded (to provide a total volume of 200 μl/well). The MTP was sealedwith tape and placed in the incubator for 30 minutes, with agitation at1400 rpm. Following incubation under the appropriate conditions, 100 μlof solution from each well were removed and placed into a fresh MTP. Thenew MTP containing 100 μl of solution/well was read at 405 nm in a MTPreader. Blank controls, as well as a control containing twomicroswatches and detergent but no enzyme were also included.

Calculation of the BMI Performance:

The obtained absorbance value was corrected for the blank value (i.e.,obtained after incubation of microswatches in the absence of enzyme).The resulting absorbance was a measure for the hydrolytic activity. Foreach sample (e.g., nprE or variant) the performance index wascalculated. The performance index compared the performance of thevariant (actual value) and the standard enzyme (theoretical value) atthe same protein concentration. In addition, the theoretical values werecalculated, using the parameters of the Langmuir equation of thestandard enzyme. A performance index (PI) that is greater than 1 (PI>1)identified a better variant (as compared to the standard [e.g.,wild-type]), while a PI of 1 (PI=1) identified a variant that performedthe same as the standard, and a PI that is less than 1 (PI<1) identifieda variant that performed worse than the standard. Thus, the PIidentified winners, as well as variants that are less desirable for useunder certain circumstances.

C. Citrate Stability Assay for NprE Protease.

Citrate stability was measured after incubation of wild-type NprE andvariants in the presence of 50 mM citrate. The initial and residualactivity was determined using the DMC hydrolysis assay. In this assaysystem, the chemical and reagent solutions used were:

Citric acid monohydrate Merck 1.00244 Pipes (free acid) Sigma P-1851Tris (free acid) Sigma T-1378 HEPES (Ultra >99.5%) Sigma-H7523TWEEN ®-80 Sigma P-8074 Dimethylcasein(DMC) Sigma C-9801 Tris buffer(free acid) 6.04 g dissolved in 1000 ml water (=50 mM) HEPES buffer 11.9g. dissolved in 1000 ml water (=50 mM) Citrate buffer (free acid) 21.0g. dissolved in 1000 ml water (=100 mM), PIPES buffer (free acid): 3.32g dissolved in about 960 ml water, DMC solution 1% w/v in 55 mM PIPESbuffer, final pH = 6.0 Dilution buffer 1 0.1 mM CaCl2/25 mM Tris; pH 8.2Dilution buffer 2 0.1 mM CaCl2/50 mM Citrate/25 mM Tris; pH 8.2

The concentrations of these dilution buffers are indicated as finalconcentrations. The initial concentration was proportionally higher anddependent on the dilution rate. The initial concentration wasproportionally higher and dependent on the dilution rate. In alternativeexperiments, HEPES finds use in exchange for Tris. The equipment usedwas a Biomek FX Robot (Beckman), and an incubator/shaker (Innova, type4230; New Brunswick). The PIPES buffer was adjusted to pH 5.8 with 4 NHCl (final concentration of 55 mM). The Tris buffer was adjusted to pH8.2 with 4 N HCl (final concentration of 25 mM). The 50 mM citrate/25 mMTris buffer was adjusted to pH 8.2 with 4 N NaOH. The HEPES buffer wasadjusted to pH 8.2 with 4 N NaOH (final concentration of 25 mM). The 50mM citrate/25 mM HEPES buffer was adjusted to pH 8.2 with 4 N NaOH.

Protein Determination

In order to establish the desired dilution rate in the citrate stabilityassay the protease concentration of the wild-type NprE controls for eachplate were determined with the TCA assay. In this method, 25 μl filteredculture broth were added to 200 μl 16.875% (w/v) TCA. After incubationfor 10 to 15 minutes at ambient temperature, the lightscattering/absorbance at 405 nm was determined. The proteinconcentration was determined by using a calibration line, constructedwith purified NprE.

Test Method

The dilution rate of the filtered culture broth was determined using theTCA assay, as described above.

Stressed Conditions:

The filtered culture broth was diluted with dilution buffer 2. The MTPwas covered with tape, shaken for a few seconds and placed in theincubator at 25° C. for 60 minutes at 200 rpm. After incubation, 20 μlof the mixture were taken from each well and transferred into a new MTP,containing 180 μl 1% DMC preheated substrate solution (the substrate waspreheated at 25° C.). The MTP was placed directly in theincubator/shaker and incubated at 25° C. for 30 minutes at 200 rpmagitation. The residual protease activity was determined using thedimethylcasein hydrolysis assay, described below.

Unstressed Conditions

The filtered culture broth was diluted with dilution buffer 1.Immediately, 20 μl of the mixture were taken from each well andtransferred into a new MTP, containing 180 μl of preheated 1% DMCsubstrate solution (the substrate was preheated at 25° C.). The MTP wasplaced directly in the incubator/shaker and incubated for 25° C. for 30minutes at 200 rpm agitation. The initial protease activity asdetermined with TNBS, using the dimethylcasein hydrolysis assay,described below.

All residual activity values (determined with the dimethylcaseinhydrolysis assay) were calculated using the following equation.% Residual Activity=OD_(60 min) value*100/OD_(00 min) valueD. Dimethylcasein Hydrolysis Assay

In this assay system, the chemicals and reagent solutions used were:

Dimethylcasein Sigma C-9801 (DMC) TWEEN ®-80 Sigma P-8074 PIPES bufferSigma P-1851; 15.1 g dissolved in about 960 ml (free acid) water; pHadjusted to 6.0 with 4N NaOH, 1 ml of 5% TWEEN ®-80 added and the volumebrought up to 1000 ml. Final concentration of PIPES and TWEEN ®-80: 50mM and 0.005% respectively. Picrylsulfonic Sigma P-2297 (5% solution inwater) acid (TNBS) Reagent A 45.4 g Na₂B₄O₇•10 H2O (Merck 6308) and 15ml of 4N NaOH dissolved together to a final volume of 1000 ml (byheating if needed) Reagent B 35.2 g NaH₂PO₄•1H₂O (Merck 6346) and 0.6 gNa₂SO₃ (Merck 6657) dissolved together to a final volume of 1000 ml.Method

To prepare the substrate, 4 g dimethylcasein was dissolved in 400 mlPIPES buffer. The filtered culture supernatants were diluted with PIPESbuffer. Then, 10 μl of each diluted supernatant were added to 200 μlsubstrate in the wells of a MTP. The MTP was covered with tape, shakenfor a few seconds and placed in an oven at 25° C. for 30 minutes withoutagitation. About 15 minutes before removal of the 1^(st) plate from theoven, the TNBS reagent was prepared by mixing 1 ml TNBS solution per 50ml of Reagent A. MTPs were filled with 60 μl TNBS Reagent A per well.The incubated plates were shaken for a few seconds, after which 10 μlwas transferred to the MTPs with TNBS Reagent A. The plates were coveredwith tape and shaken for 20 minutes in a bench shaker (BMG Thermostar)at room temperature and 500 rpm. Finally, 200 μl Reagent B was added tothe wells, mixed for 1 minute on a shaker, and the absorbance at 405 nmwas determined using a MTP reader.

The obtained absorbance value was corrected for the blank value (i.e.,substrate without enzyme). The resulting absorbance was a measure of thehydrolytic activity. The (arbitrary) specific activity of a sample wascalculated by dividing the absorbance and the determined proteinconcentration.

E. TIDE® Stability Assay

The stability of NprE and variants was measured after an incubation stepin the presence of 25% TIDE® compact detergent. The initial and residualactivity was determined using the AGLA-assay described below. Theequipment used was a Biomek FX Robot (Beckman), a fluorescence meter(FLUOstar Optima; BMG), an incubator/shaker (iEMS; Thermoelectron) andan incubator/shaker (Innova; New Brunswick (type 4230)); the MTPs werefrom Costar (type 9017) and from Greiner (black plates, type 655076).

Chemicals and Reagents:

In this assay system, the chemical and reagent solutions used were:

TIDE ®-compact With and without DTPA detergent TIDE ®-compact 125 gTIDE ®-compact dissolved in a mixture of 50 g detergent solution of 50mM HEPES pH 8.2 and 275 ml water; concentration of TIDE ® was 27.7%,after dilution with supernatant 25% MES dilution 52.6 mM MES/NaOH, 2.6mM CaCl₂, 0.005% buffer TWEEN ®-80, pH 6.5 AGLA substrate BaChem, catno. H-6675 or American Peptide Company, cat no. 81-0-31 AGLA substrate451 mg of AGLA dissolved in 16 ml N,N- solution dimethylformamide; thissolution was poured into 304 ml of MES-buffer (52.6 mM MES/NaOH, 2.6 mMCaCl₂, 0.005% TWEEN ®-80, pH 6.5) with stirringTest Method:Unstressed Conditions:

First, 20 μl filtered culture broth was diluted with 180 μl MES dilutionbuffer. Then, 20 μl of this diluted broth was diluted with 180 μl MESdilution buffer. Then, 10 μl of this dilution was diluted with 190 μlAGLA-substrate solution in a pre-warmed plate at 25° C. Any air bubblespresent were blown away and the plate was measured according to the AGLAprotease assay protocol.

Stressed Conditions:

First, 20 μl filtered culture broth was diluted with 180 μlTIDE®-compact detergent solution without DTPA and after premixing in theiEMS shaker for 5 minutes, were incubated further in the Innova shaker.The plate was incubated for a total of 60 minutes at 32° C., at 200 rpm.In addition, 20 ul filtered culture broth were diluted with 180 ulTIDE®-compact detergent solution with DTPA and after premixing in theiEMS shaker for 5 minutes, were incubated further in the Innova shaker.The plate was incubated for a total of 40 minutes at 20° C., at 200 rpm.Then, 20 μl of either of these solutions were diluted with 180 μl MESdilution buffer and 10 μl of this dilution were diluted with 190 μlAGLA-substrate solution in a pre-warmed plate at 25° C. Any air bubblespresent were blown away and the plate was measured according to the AGLAprotease assay protocol.

Calculations:

Fluorescence measurements were taken at excitation of 350 nm andemission of 415 nm. The spectrofluorometer software calculated thereaction rates of the increase in fluorescence for each well to alinearly regressed line of milli-RFU/min:

${Percentage}\mspace{14mu}{of}\mspace{14mu}{residual}\mspace{14mu}{activity}\text{:}\mspace{14mu}\frac{\left( {{Slope}\mspace{14mu}{of}\mspace{14mu}{stressed}\mspace{14mu}{condition}} \right) \star 100}{\left( {{Slope}\mspace{14mu}{of}\mspace{14mu}{unstressed}\mspace{14mu}{condition}} \right)}$F. 2-Aminobenzoyl-L-alanylgycyl-L-leucyl-L-alamino-4-nitrobenzylamideProtease Assay (Abz-AGLA-Nba)

The method provided below provides a degree of technical detail thatyields reproducible protease assay data independent of time and place.While the assay can be adapted to a given laboratory condition, any dataobtained through a modified procedure must be reconciled with resultsproduced by the original method. Neutral metalloproteases cleave thepeptide bond between glycine and leucine of2-aminobenzoyl-L-alanylglycyl-L-leucyl-L-alamino-4-nitrobenzylamide(Abz-AGLA-Nba). Free 2-aminobenzoyl-L-alanylglycine (Abz-AG) in solutionhas a fluorescence emission maximum at 415 nm with an excitation maximumof 340 nm. Fluorescence of Abz-AG is quenched by nitrobenzylamide in theintact Abz-AGLA-Nba molecule.

In these experiments, the liberation of Abz-AG by protease cleavage ofAbz-AGLA-Nba was monitored by fluorescence spectroscopy (Ex. 340/Em.415). The rate of appearance of Abz-AG was a measure of proteolyticactivity. Assays were performed under non-substrate limited initial rateconditions.

A microplate mixer with temperature control (e.g., EppendorfThermomixer) was required for reproducible assay results. The assaysolutions were incubated to desired temperature (e.g., 25° C.) in themicroplate mixer prior to enzyme addition. Enzyme solutions were addedto the plate in the mixer, mixed vigorously and rapidly transferred tothe plate reader.

A spectrofluorometer with capability of continuous data recording,linear regression analysis, and with temperature control was required(e.g., SpectraMax M5, Gemini EM, Molecular Devices). The reader wasalways maintained at the desired temperature (e.g., 25° C.). The readerwas set for top-read fluorescence detection and the excitation was setto 350 nm and emission to 415 nm without the use of a cut-off filter.The PMT was set to medium sensitivity and 5 readings per well.Autocalibration was turned on, but only to calibrate before the firstreading. The assay was measured for 3 minutes with the reading intervalminimized according to the number of wells selected to be monitored. Thereader was set to calculate the rate of milli-RFU/min (thousandths ofrelative fluorescence units per minute). The number of readings used tocalculate the rate (Vmax points) was set to the number equivalent to 2minutes, as determined by the reading interval (e.g., a reading every 10seconds would use 12 points to calculate the rate). The max RFU was setto 50,000.

All pipeting of enzyme and substrate stock solutions were done withpositive displacement pipets (Rainin Microman). Buffer, assay, andenzyme working solutions were pipetted by single or multi-channelair-displacement pipets (Rainin LTS) from tubes, reagent reservoirs orstock microplates. A repeater pipet (Eppendorf) finds use intransferring the assay solution to microplate wells when few wells areused, to minimize reagent loss. Automated pipetting instruments such asthe Beckman FX or Cybio Cybi-well also find use in transferring enzymesolutions from a working stock microplate to the assay microplate inorder to initiate an entire microplate at once.

Reagents and Solutions:

52.6 mM MES/NaOH, 2.6 mM CaCl₂, pH 6.5—MES Buffer

MES acid (10.28 g) and 292 mg anhydrous CaCl₂ were dissolved inapproximately 900 mL purified water. The solution was titrated with NaOHto pH 6.5 (at 25° C. or with temperature adjustment pH probe). ThepH-adjusted buffer was made up to IL total volume. The final solutionwas filtered through a 0.22 μm sterile filter and kept at roomtemperature.

48 mM Abz-AGLA-Nba in DMF—Abz-AGLA-Nba Stock

Approximately 28 mg of Abz-AGLA-Nba was placed in a small tube. It wasdissolved in mL of DMF (volume will vary depending upon Abz-AGLA-Nbamassed) and vortexed for several minutes. The solution was stored atroom temperature shielded from light.

50 mM MES, 2.5 mM CaCl₂, 5% DMF, 2.4 mM Abz-AGLA-Nba pH 6.5—AssaySolution

One mL Abz-AGLA-Nba stock was added to 19 mL MES Buffer and vortexed.The solution was stored at room temperature shielded from light.

50 mM MES, 2.5 mM CaCl₂, pH 6.5—Enzyme Dilution Buffer

This buffer was produced by adding 5 mL purified water to 95 mL MESBuffer.

50 mM MES, 2.5 mM CaCl₂, 5% DMF, pH 6.5—Substrate Dilution Buffer

Five mL pure DMF were added to 95 mL MES Buffer. This buffer was used todetermine kinetic parameters.

Enzyme Solutions

The enzyme stock solutions were diluted with enzyme dilution buffer to aconcentration of approximately 1 ppm (1 ug/mL). MULTIFECT® neutralprotease (wild-type NprE) was diluted to concentrations below 6 ppm (6ug/mL). Serial dilutions were preferred. Solutions were stable at roomtemperature for 1 hour, but for longer term storage, the solutions weremaintained on ice.

Procedure

First all buffers, stock, and working solutions were prepared. Eachenzyme dilution was assayed in triplicate, unless otherwise indicated.When not completely full, the enzyme working solution stock microplatewas arranged in full vertical columns starting from the left of theplate (to accommodate the plate reader). The corresponding assay platewas similarly set up. The microplate spectrofluorometer was set up aspreviously described.

First, a 200 uL aliquot of assay solution were placed in the wells of a96-well microplate. The plate was incubated for 10 min at 25° C. in atemperature controlled microplate mixer, shielded from light. The assaywas initiated by transferring 10 uL of the working enzyme solutions fromthe stock microplate to the assay microplate in the mixer. Optimally,96-well pipetting head finds use, or an 8-well multi-channel pipet wasused to transfer from the left-most column first. The solutions werevigorously mixed for 15 seconds (900 rpm in Eppendorf Thermomixer).Immediately, the assay microplate was transferred to the microplatespectrofluorometer and recording of fluorescence measurements atexcitation of 350 nm and emission of 415 nm were begun. Thespectrofluorometer software calculated the reaction rates of theincrease in fluorescence for each well to a linearly regressed line ofmilli-RFU/min. In some experiments, a second plate was placed in themicroplate mixer for temperature equilibration while the first plate wasbeing read.

The rate initial velocities were linear with respect to productconcentration (i.e., liberated 2-aminobenzoyl fluorescence) up to 0.3 mMproduct, which corresponded to approximately 50,000 RFU in a solutionstarting at 2.3 mM Abz-AGLA-Nba with background fluorescence ofapproximately 22,000 RFU. Abz-AGLA-Nba was dissolved in DMF and was beenused the day it was prepared.

Detergent Compositions:

In the exemplified detergent compositions, the enzymes levels areexpressed by pure enzyme by weight of the total composition and unlessotherwise specified, the detergent ingredients are expressed by weightof the total compositions. The abbreviated component identificationstherein have the following meanings:

Abbreviation Ingredient LAS Sodium linear C₁₁₋₁₃ alkyl benzenesulfonate. NaC16-17HSAS Sodium C₁₆₋₁₇ highly soluble alkyl sulfate TASSodium tallow alkyl sulphate. CxyAS Sodium C_(1x)-C_(1y) alkyl sulfate.CxyEz C_(1x)-C_(1y) predominantly linear primary alcohol condensed withan average of z moles of ethylene oxide. CxyAEzS C_(1x)-C_(1y) sodiumalkyl sulfate condensed with an average of z moles of ethylene oxide.Added molecule name in the examples. Nonionic Mixedethoxylated/propoxylated fatty alcohol e.g. Plurafac LF404 being analcohol with an average degree of ethoxylation of 3.8 and an averagedegree of propoxylation of 4.5. QAS R₂•N+(CH₃)₂(C₂H₄OH) with R₂ =C₁₂-C₁₄. Silicate Amorphous Sodium Silicate (SiO₂:Na₂O ratio =1.6-3.2:1). Metasilicate Sodium metasilicate (SiO₂:Na₂O ratio = 1.0).Zeolite A Hydrated Aluminosilicate of formula Na₁₂(A1O₂SiO₂)₁₂•27H₂OSKS-6 Crystalline layered silicate of formula δ-Na₂Si₂O_(5.) SulfateAnhydrous sodium sulphate. STPP Sodium Tripolyphosphate. MA/AA Randomcopolymer of 4:1 acrylate/maleate, average molecular weight about70,000-80,000. AA Sodium polyacrylate polymer of average molecularweight 4,500. Polycarboxylate Copolymer comprising mixture ofcarboxylated monomers such as acrylate, maleate and methyacrylate with aMW ranging between 2,000-80,000 such as Sokolan commercially availablefrom BASF, being a copolymer of acrylic acid, MW4,500. BB13-(3,4-Dihydroisoquinolinium)propane sulfonate BB21-(3,4-dihydroisoquinolinium)-decane-2-sulfate PB1 Sodium perboratemonohydrate. PB4 Sodium perborate tetrahydrate of nominal formulaNaBO₃•4H₂O. Percarbonate Sodium percarbonate of nominal formula2Na₂CO₃•3H₂O₂. TAED Tetraacetyl ethylene diamine. NOBSNonanoyloxybenzene sulfonate in the form of the sodium salt. DTPADiethylene triamine pentaacetic acid. HEDP 1,1-hydroxyethanediphosphonic acid. DETPMP Diethyltriamine penta (methylene) phosphonate,marketed by Monsanto under the Trade name Dequest 2060. EDDSEthylenediamine-N,N′-disuccinic acid, (S,S) isomer in the form of itssodium salt Diamine Dimethyl aminopropyl amine; 1,6-hezane diamine;1,3-propane diamine; 2-methyl-1,5-pentane diamine; 1,3-pentanediamine;1- methyl-diaminopropane. DETBCHD 5,12-diethyl-1,5,8,12-tetraazabicyclo[6,6,2] hexadecane, dichloride, Mn(II) SALT PAAC Pentaamine acetatecobalt(III) salt. Paraffin Paraffin oil sold under the tradename Winog70 by Wintershall. Paraffin Sulfonate A Paraffin oil or wax in whichsome of the hydrogen atoms have been replaced by sulfonate groups.Aldose oxidase Oxidase enzyme sold under the tradename Aldose Oxidase byNovozymes A/S Galactose oxidase Galactose oxidase from Sigma nprE Therecombinant form of neutral metalloprotease expressed in Bacillussubtilis. PMN Purified neutral metalloprotease from Bacillusamyloliquefacients. Amylase Amylolytic enzyme sold under the tradenamePURAFECT ® Ox described in WO 94/18314, WO96/05295 sold by Genencor;NATALASE ®, TERMAMYL ®, FUNGAMYl ® and DURAMYL ™, all available fromNovozymes A/S. Lipase Lipolytic enzyme sold under the tradenameLIPOLASE ®, LIPOLASE ® Ultra by Novozymes A/S and Lipomax ™ by Gist-Brocades. Cellulase Cellulytic enzyme sold under the tradename Carezyme,Celluzyme and/or Endolase by Novozymes A/S. Pectin Lyase PECTAWAY ® andPECTAWASH ® available from Novozymes A/S. PVP Polyvinylpyrrolidone withan average molecular weight of 60,000 PVNO Polyvinylpyridine-N-Oxide,with an average molecular weight of 50,000. PVPVI Copolymer ofvinylimidazole and vinylpyrrolidone, with an average molecular weight of20,000. Brightener 1 Disodium 4,4′-bis(2-sulphostyryl)biphenyl. Siliconeantifoam Polydimethylsiloxane foam controller with siloxane-oxyalkylenecopolymer as dispersing agent with a ratio of said foam controller tosaid dispersing agent of 10:1 to 100:1. Suds Suppressor 12%Silicone/silica, 18% stearyl alcohol, 70% starch in granular form. SRP 1Anionically end capped poly esters. PEG X Polyethylene glycol, of amolecular weight of x. PVP K60 ® Vinylpyrrolidone homopolymer (averageMW 160,000) Jeffamine ® ED-2001 Capped polyethylene glycol from HuntsmanIsachem ® AS A branched alcohol alkyl sulphate from Enichem MME PEG(2000) Monomethyl ether polyethylene glycol (MW 2000) from Fluka ChemieAG. DC3225C Silicone suds suppresser, mixture of Silicone oil and Silicafrom Dow Corning. TEPAE Tetreaethylenepentaamine ethoxylate. BTABenzotriazole. Betaine (CH₃)₃N⁺CH₂COO⁻ Sugar Industry grade D-glucose orfood grade sugar CFAA C₁₂-C₁₄ alkyl N-methyl glucamide TPKFA C₁₂-C₁₄topped whole cut fatty acids. Clay A hydrated aluminumu silicate in ageneral formula Al₂O₃SiO₂•xH₂O. Types: Kaolinite, montmorillonite,atapulgite, illite, bentonite, halloysite. pH Measured as a 1% solutionin distilled water at 20° C.

Example 1 Cloning of the Neutral Metalloprotease Gene from B.amyloliquefaciens

In this Example, methods used to clone the B. amyloliquefaciens neutralmetalloprotease gene are described. The gene-encoding neutralmetalloprotease was cloned from B. amyloliquefaciens usingwell-established methods in this art. The non-exempt (i.e., the straincarries extrageneric DNA (besides the chloramphenicol selectable markerwhich is allowed in an exempt strain), specifically the plasmid pJM102sequences) strain BC91504 (aprE/nprE-pJM102 in BG3594::comK) carries theB. subtilis aprE promoter and signal sequence fused to B.amyloliquefaciens nprE propeptide/mature gene in integrating plasmidpJM102.

The following two sequences (SEQ ID NO:1 and SEQ ID NO:2) of B. subtilisand B. amyloliquefaciens were generated via PCR with the oligonucleotideprimers corresponding to the underlined sequences.

B subtilis chromosomal EcoRI restriction site (GAATTC) and aprE startcodon (GTG) and B. amyloliquefaciens nprE stop codon are shown in thefollowing sequences in boldface type as well as a syntheticallyintroduced HindIII restriction site (AAGCTT) designed into primer #4.

The B. amyloliquefaciens aprE 5′ upstream sequence, promoter and signalsequence coding region are shown in the following sequence (SEQ IDNO:1). Primer 1 (apr-f; GAGCTGGGTAAAGCCTATGAAT; SEQ ID NO:5) is shownunderlined, at the beginning of the sequence, while the aprE portion ofprimers 2 and 3 (npr-f and npr-r; GTTCAGCAACATGTCTGCGCAGGCT; SEQ IDNO:6) are shown double underlined at the end of the sequence.

(SEQ ID NO: 1) GAGCTGGGTAAAGCCTATGAAT TCTCCATTTTCTTCTGCTATCAAAATAACAGACTCGTGATTTTCCAAACGAGCTTTCAAAAAAGCCTCTGCCCCTTGCAAATCGGATGCCTGTCTATAAAATTCCCGATATTGGTTAAACAGCGGCGCAATGGCGGCCGCATCTGATGTCTTTGCTTGGCGAATGTTCATCTTATTTCTTCCTCCCTCTCAATAATTTTTTCATTCTATCCCTTTTCTGTAAAGTTTATTTTTCAGAATACTTTTATCATCATGCTTTGAAAAAATATCACGATAATATCCATTGTTCTCACGGAAGCACACGCAGGTCATTTGAACGAATTTTTTCGACAGGAATTTGCCGGGACTCAGGAGCATTTAACCTAAAAAAGCATGACATTTCAGCATAATGAACATTTACTCATGTCTATTTTCGTTCTTTTCTGTATGAAAATAGTTATTTCGAGTCTCTACGGAAATAGCGAGAGATGATATACCTAAATAGAGATAAAATCATCTCAAAAAAATGGGTCTACTAAAATATTATTCCATCTATTACAATAAATTCACAGAATAGTCTTTTAAGTAAGTCTACTCTGAATTTTTTTAAAAGGAGAGGGTAAAGAGTGAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAACGTTAATCTTTACGATGGCGTTCAGCAACAT GTCTGCGCAGGCT

The sequence of the B. amyloliquefaciens propeptide and mature nprEcoding sequence and transcription terminator are provided in thesequence below. In this sequence, the nprE portion of primers 2 and 3 isunderlined (GCTGAGAATCCTCAGCTTAAAGAAAACCTG; SEQ ID NO:7), while thenpr-r portion of primer 4 (GGCTTCACCATGATCATATATGTCAAGCTTGGGGGG; SEQ IDNO:8) is shown double underlined.

(SEQ ID NO: 2) GCTGAGAATCCTCAGCTTAAAGAAAACCTGACGAATTTTGTACCGAAGCATTCTTTGGTGCAATCAGAATTGCCTTCTGTCAGTGACAAAGCTATCAAGCAATACTTGAAACAAAACGGCAAAGTCTTTAAAGGCAATCCTTCTGAAAGATTGAAGCTGATTGACCAAACGACCGATGATCTCGGCTACAAGCACTTCCGTTATGTGCCTGTCGTAAACGGTGTGCCTGTGAAAGACTCTCAAGTCATTATTCACGTCGATAAATCCAACAACGTCTATGCGATTAACGGTGAATTAAACAACGATGTTTCCGCCAAAACGGCAAACAGCAAAAAATTATCTGCAAATCAGGCGCTGGATCATGCTTATAAAGCGATCGGCAAATCACCTGAAGCCGTTTCTAACGGAACCGTTGCAAACAAAAACAAAGCCGAGCTGAAAGCAGCAGCCACAAAAGACGGCAAATACCGCCTCGCCTATGATGTAACCATCCGCTACATCGAACCGGAACCTGCAAACTGGGAAGTAACCGTTGATGCGGAAACAGGAAAAATCCTGAAAAAGCAAAACAAAGTGGAGCATGCCGCCACAACCGGAACAGGTACGACTCTTAAAGGAAAAACGGTCTCATTAAATATTTCTTCTGAAAGCGGCAAATATGTGCTGCGCGATCTTTCTAAACCTACCGGAACACAAATTATTACGTACGATCTGCAAAACCGCGAGTATAACCTGCCGGGCACACTCGTATCCAGCACCACAAACCAGTTTACAACTTCTTCTCAGCGCGCTGCCGTTGATGCGCATTACAACCTCGGCAAAGTGTATGATTATTTCTATCAGAAGTTTAATCGCAACAGCTACGACAATAAAGGCGGCAAGATCGTATCCTCCGTTCATTACGGCAGCAGATACAATAACGCAGCCTGGATCGGCGACCAAATGATTTACGGTGACGGCGACGGTTCATTCTTCTCACCTCTTTCCGGTTCAATGGACGTAACCGCTCATGAAATGACACATGGCGTTACACAGGAAACAGCCAACCTGAACTACGAAAATCAGCCGGGCGCTTTAAACGAATCCTTCTCTGATGTATTCGGGTACTTCAACGATACTGAGGACTGGGATATCGGTGAAGATATTACGGTCAGCCAGCCGGCTCTCCGCAGCTTATCCAATCCGACAAAATACGGACAGCCTGATAATTTCAAAAATTACAAAAACCTTCCGAACACTGATGCCGGCGACTACGGCGGCGTGCATACAAACAGCGGAATCCCGAACAAAGCCGCTTACAATACGATTACAAAAATCGGCGTGAACAAAGCGGAGCAGATTTACTATCGTGCTCTGACGGTATACCTCACTCCGTCATCAACTTTTAAAGATGCAAAAGCCGCTTTGATTCAATCTGCGCGGGACCTTTACGGCTCTCAAGATGCTGCAAGCGTAGAAGCTGCCTGGAATGCAGTCGGATTGTAAACAAGAAAAGAGACCGGAAATCCGGTCTCTTTTTTATATCTAAAAACATTTCACAGTGGCTTCAC CATGATCATATATGTCAAGCTT GGGGGG

The amino acid sequence of the full-length NprE (pre-, pro- and maturesequence) is provided below:

(SEQ ID NO: 3) MGLGKKLSVAVAASFMSLTISLPGVQAAENPQLKENLTNFVPKHSLVQSELPSVSDKAIKQYLKQNGKVKGNPSERLKLIDQTTDDLGYKHFRYVPVVNGVPVKDSQVIIHVDKSNNVYAINGELNNDVSAKTANSKKLSANQALDHAYKAIGKSPEAVSNGTVANKNKAELKAAATKDGKYRLAYDVTIRYIEPEPANWEVTVDAETGKILKKQNKVEHAATTGTGTTLKGKTVSLNISSESGKYVLRDLSKPTGTQIITYDLQNREYNLPGTLVSSTTNQFTTSSQRAAVDAHYNLGKVYDYFYQKFNRNSYDNKGGKIVSSVHYGSRYNNAAWIGDQMIYGDGDGSFFSPLSGSMDVTAHEMTHGVTQETANLNYENQPGALNESFSDVFGYFNDTEDWDIGEDITVSQPALRSLSNPTKYGQPDNFKNYKNLPNTDAGDYGGVHTNSGIPNKAAYNTITKIGVNKAEQIYYRALTVYLTPSSTFKDAKAALIQSAR DLYGSQDAASVEAAWNAVGL

In some alternative embodiments, the following NprE sequence finds usein the present invention.

(SEQ ID NO: 4) VRSKKLWISLLFALTLIFTMAFSNMSAQAAENPQLKENLTNFVPKHSLVQSELPSVSDKAIKQYLKQNGKVFKGNPSERLKLIDQTTDDLGYKHFRYVPVVNGVPVKDSQVIIHVDKSNNVYAINGELNNDVSAKTANSKKLSANQALDHAYKAIGKSPEAVSNGTVANKNKAELKAAATKDGKYRLAYDVTIRYIEPEPANWEVTVDAETGKILKKQNKVEHAATTGTGTTLKGKTVSLNISSESGKYVLRDLSKPTGTQIITYDLQNREYNLPGTLVSSTTNQFTTSSQRAAVDAHYNLGKVYDYFYQKFNRNSYDNKGGKIVSSVHYGSRYNNAAWIGDQMIYGDGDGSFFSPLSGSMDVTAHEMTHGVTQETANLNYENQPGALNESFSDVFGYFNDTEDWDIGEDITVSQPALRSLSNPTKYGQPDNFKNYKNLPNTDAGDYGGVHTNSGIPNKAAYNTITKIGVNKAEQIYYRALTVYLTPSSTFKDAKAALIQSARDLYGSQDAASVEAAWNAVGL

The primer sequences used in these PCR experiments are provided below:

Primers Used in PCR Experiments SEQ Primer ID Number Sequence NO: 15′-GAGCTGGGTAAAGCCTATGAAT-3′ SEQ ID NO: 5 25′-CAGGTTTTCTTTAAGCTGAGGATTCTCAGC- SEQ ID AGCCTGCGCAGACATGTTGCTGAAC-3′NO: 9 3 5′-GTTCAGCAACATGTCTGCGCAGGCT- SEQ IDGCTGAGAATCCTCAGCTTAAAGAAAACCTG-3′ NO: 10 45′-CCCCCCAAGCTTGACATATATGATCATGGTGA- SEQ ID AGCC-3′ NO: 11

Primers 2 and 3 are reverse complements of each other and correspond toeither non-coding (#2) or coding (#3) strands of the chromosomal DNAs.For the coding strand, they correspond to the last 25 base pairs of theaprE signal sequence and the first 30 base pairs of the nprE propeptide.Primer #4 is the reverse complement to the underlined sequence,comprising 24 base pairs 3′ of the nprE stop codon and terminator withan introduced HindIII site preceded by six dCTP residues, to provide aso-called “clamp,” allowing more efficient cleavage with HindIIIrestriction endonuclease, as some restriction enzymes cleaveinefficiently if their recognition sequence is located at the very endsof DNA fragments.

The two PCR fragments were generated with the following protocol andreagents (except DNA template and oligonucleotide primers) from AppliedBiosystems' rTTH DNA Polymerase, XL Kit:

40.6 μl H₂O

30 μl 3.3× rTth PCR buffer

10 μl 2 mM dNTP mix

4.4 μl 25 mM Mg-acetate

5 μl 50 μM primer #1 or #3 (forward primers)

5 μl 50 μM primer #2 or #4 (reverse primers)

2 μl B. subtilis or B. amyloliquefaciens chromosomal DNA

2 μl rTth polymerase

1 μl Pfu Turbo polymerase

100 μl total reaction volume

The PCR conditions used in these experiments were (95° C., 30 sec./58°C., 30 sec/68° C., 1 min.)×30 cycles followed by rapid cooling to 4° C.Reactions were run on 1.2% agarose/TBE preparative gels, theappropriately-sized fragments excised and purified using the QIAGEN® GelExtraction Kit. In a second fusion, PCR reactions were conducted inwhich chromosomal DNAs were replaced by 1 ul each of the two separatefragments and only outside primers #s 1 and #2 were used. The same PCRconditions as described above were used. Due to the complementary endsformed on the two fragments from the use of complementary primers 2 and3 in the first PCRs, the two fragments were precisely fused.

The fusion fragment was digested with EcoRI and HindIII and gel purifiedas described above. The integration plasmid pJM102 was also digestedwith EcoRI and HindIII, and the linear plasmid was then gel purified andligated by standard techniques to the digested apr/npr fusion fragment.This ligation reaction was subsequently used to directly transform axylose-induced B. subtilis strain.

After purification, the two fragments were generated by PCR with primers1 and 2 from wild-type B. subtilis chromosomal DNA, and with primers 3and 4 from chromosomal DNA from a B. amyloliquefaciens strain. Thisfragment was again purified as descried above, followed by cutting withEcoRI and HindIII as in the same digestion of the integrating plasmidpJM102 and subsequent ligation of the fusion fragment to the plasmid.Several transformants had the fusion sequenced from the chromosome toverify the absence of any PCR-derived mutations. One of these was thenamplified stepwise from 5-25 mg/mL chloramphenicol, the selectablemarker on pJM102, to co-amplify the linked expression cassette.

The selected sequence verified transformant was obtained by selectionfor pJM102's chloramphenicol (CMP) resistance marker on LB/agar platescontaining 5 mg/ml CMP. This was then inoculated into LB broth at 10mg/ml CMP overnight at 37° C., with shaking at 250 RPM. This culture wasthen streaked onto LB/agar plates with 10 mg/ml CMP to isolate singlecolonies. One colony was then inoculated into LB broth at 25 mg/ml CMPovernight at 37° C., with shaking at 250 RPM. This culture was thenstreaked to LB/agar plates with 25 mg/ml CMP to isolate single colonies.These colonies were harvested and stored in glycerol at −70° C. untiluse, as known in the art.

The deletion of the two non-essential proteases present in B. subtilis(aprE and nprE), as well as amylase, reduced the total extracellularprotease level during the production of metalloprotease. The DNAencoding the neutral metalloprotease was cloned into an amylase-deletedhost. The inducible comK for competence development was inserted in themiddle of the amylase locus, making the strain “amy⁻.” The secretion ofthe expressed protein was ensured by insertion of the nucleotidesencoding the signal sequence prior to the coding sequence of the gene.

Example 2 Expression and Fermentation of the Purified MULTIFECT® Neutraland Recombinant Neutral Metalloprotease (nprE)

The recombinant Bacillus subtilis produced as described in Example 1 wascultivated by conventional batch fermentation in a nutrient medium asdescribed below. One glycerol vial (prepared as described in Example 1)of B. subtilis culture containing the B. amyloliquefaciens neutralmetalloprotease was used to inoculate 600 ml of SBG1% medium containing200 mg/L chloramphenicol. The cultures were grown for 48 hours at 37°C., after which time, the culture fluid was recovered by centrifugationat 12,000 rpm, as known in the art. This procedure was done induplicate. The final enzyme concentrations obtained were in the range ofabout 1.4 and 2 g/L.

Example 3 Purification and Characterization of Neutral Metalloprotease

This Example describes the methods used to purify the neutralmetalloprotease expressed by the organisms described in Example 2. After36 hours of incubation at 37° C., the fermentation broth was recoveredand centrifuged at 12 000 rpm (SORVALL® centrifuge model RC5B). Thesecreted neutral metalloproteases were isolated from the culture fluidand concentrated approximately 10-fold using an Amicon filter system8400 with a BES (polyethersulfone) 10 kDa cutoff.

The concentrated supernatant was dialyzed overnight at 4° C. against 25mM MES buffer, pH 5.4, containing 10 mM NaCl. The dialysate was thenloaded onto a cation-exchange column Porous HS20 (total volume ˜83 mL;binding capacity ˜4.5 g protein/mL column; Waters) as described below.The column was pre-equilibrated with 25 mM MES buffer, pH 5.4,containing 10 mM NaCl. Then, approximately 200-300 mL of sample wasloaded onto the column. The bound protein was eluted using a pH gradientfrom 5.4 to 6.2 over 10-column volumes of MES buffer. Elution of theprotein was between pH 5.82 and 6.0, and was assessed using proteolyticactivity as described herein and 10% (w/v) NUPAGE® SDS-PAGE (Novex). Theneutral protease containing fractions were then pooled. Calcium and zincchloride salts in the ratio of 3:1 were added prior to the adjustment ofthe pH to 5.8. The Perceptive Biosystems BIOCAD® Vision (GMI) was usedfor protein purification.

The purified protein, assessed using a 10% (w/v) NUPAGE® SDS-PAGE, wasdetermined to homogenous, with greater than 95% purity. Typically, lessthan 1% of the purified preparations showed serine protease activitywhen assessed using the standard protease assay with the smallsubstrate, suc-p-AAPF-pNA(N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide) (Sigma). This assaywas performed in microtiter plate format (96 well) using a 100 mMTris-HCl buffer, pH 8.5, containing 10 mM CaCl₂ and 0.005% TWEEN®-80.The substrate (p-AAPF NA) was prepared by making a 160 mM stock in DMSO(dimethylsulfoxide) (100 mg/ml) and diluting this stock 100-fold withthe Tris-HCl buffer containing CaCl₂ and 0.005% TWEEN®-80. Then, 10 uLof diluted protease solution (dilutions were prepared using 100 mMTris-HCl buffer, pH 8.5, containing 10 mM CaCl₂ and 0.005% TWEEN-80) wasadded to 190 uL 1 mg/ml p-AAPF solution. The assay was mixed for 5minutes and the kinetic change at 410 nm was read over 2 to 5 minutes.The slope of the response was measured and used as an indication of theamount of serine protease, activity. The protein was formulated forstorage using 25 mM MES buffer, pH 5.8, containing 1 mM zinc chloride, 4mM calcium chloride, and 40% propylene glycol.

Example 4 Affinity of Purified MULTIFECT® Neutral Metalloprotease (PMN)for Calcium and Zinc Cations

In this Example, methods to determine the affinity of the neutralmetalloprotease (PMN) prepared as described in the above Examples aredescribed. The affinities of PMN for calcium and zinc ions wereperformed using the fluorescent indicators Fluo-3 and FluoZin-3,respectively obtained from Molecular Probes. All fluorescencemeasurements were recorded on a LS50B Luminescence spectrophotometer(Perkin-Elmer). The binding of Fluo-3 was monitored by excitation at 500nm and the emission spectra were recorded from 505 to 550 nm. Similarly,the binding of FluoZin-3 was monitored by excitation at 495 nm and theemission spectra were collected from 500 to 550 nm. The excitation andemission slit width were both set at 2.5 nm.

In these determinations, 100 uM neutral metalloprotease in 50 mMTris-HCl buffer, pH 8.4, was titrated with increasing amounts of therelevant indicator. The titration curves are shown in FIG. 1. In thisFigure, the triangles represent the curve binding data obtained forZn²⁺, using the Fluo-Zin3 dye monitored at 516 nm, while the circlesrepresent the data obtained for Ca²⁺ using the Fluo-3 dye monitored at522 nm. The association constants (Ka's) for zinc and calcium (assuminga single binding site) were determined to be 0.401 nM and 0.037 nM,respectively. These results indicate that purified MULTIFECT® neutralmetalloprotease bound the zinc ion with approximately 10-fold greateraffinity than the calcium ion. Based on the weaker binding of calcium,initial protein engineering experiments are designed to involve either(i) designing tighter calcium binding site(s) and/or (ii) eliminatingthe structural stability requirement for calcium (e.g., to stabilize theprotein to greater than 80%).

Example 5 Storage Stability

In this Example, experiments conducted to assess the storage stabilityof PMN and recombinant B. amyloliquefaciens neutral metalloproteaseexpressed in B. subtilis are described. Proteolysis of these neutralmetalloprotease preparations was assessed in the presence of increasingLAS (lauryl sodium sulfate; Sigma) solutions (0% up to an including10%). Proteolytic fragments generated from the purified MULTIFECT®neutral metalloprotease (PMN) were observed using 10% (w/v) NUPAGE®SDS-PAGE.

The storage stability of the recombinant neutral metalloprotease from B.amyloliquefaciens expressed in B. subtilis produced as described above,was determined in buffer alone (50 mM Tris-HCl buffer, pH 8.4) and inthe presence of detergent base obtained from Procter & Gamble. Thebuffer and/or detergent base contained zinc ions, calcium ions or acombination thereof. The concentration of both the zinc and calcium ionswas varied from 0 to 25 mM. These results were always compared withthose for the neutral metalloprotease incubated in buffer alone.

Protease Assays

Azo-Casein Assay:

The azo-casein endpoint assay was used to assess the amount ofproteolysis that occurred under certain conditions. In these assays, 75uL of enzyme were incubated with excess calcium or zinc or both ionsadded to 250 μl of 1% (w/v) azo-casein (Sigma). The reaction proceededat 30° C. for 15 minutes, after which 10% (w/v) trichloroacetic acid wasadded to stop the reaction. The precipitated protein and the unreactedazo-casein were removed by centrifugation for 10 minutes at 14 000 rpm.The color of the azo-group was developed by addition of 750 μL 1 Msodium hydroxide. The development of the color proceeded for 5 minutes,after which the reaction was stopped and the absorbance was measured at440 nm.

Succinylated-Casein and TNBSA Assay:

The activity of the neutral metalloprotease was determined using theQuantiCleave Protease Assay Kit™ (Pierce). This assay is based on thedigestion of succinylated-casein by the enzyme. The primary amino groupsformed are then reacted with trinitrobenzene sulfonic acid (TNBSA) andform a colored complex that has maximum absorbance at 450 nm. The assayis performed in 96-well microtiter format. The assay requires a15-minute incubation with the succinylated casein and a 15-minutereaction with the TNBSA. During both incubations, the samples are placedon a shaker. TPCK-trypsin (Pierce) is the general standard used foroverall protease activity determinations. However, optimum conditionsfor activity for specific proteases require the use of the protease ofinterest. In the case of the assays performed in these experiments, bothtrypsin and the protease of interest were used, in order to calibratethe assay. The accuracy of the assay requires that the standarddilutions made of 0.5 mg/mL trypsin always result in absorbance values(at 450 nm) below 0.5.

Every sample was measured relative to a control containing no casein.The reported change in absorbance (ΔAbs(450 nm)) accounts for theinterference from the amino groups of casein. Further, any possibleinterference from primary amino groups in the buffer and/or othercomponents of the detergent was/were also corrected for in this manner.The activity of all samples was determined relative to detergent with noadded neutral metalloprotease, as well as for enzyme incubated in BupH™borate buffer supplied with the kit, for the same length of time and atthe same temperature.

This test is an end-point assay, in which 50 mM borate buffer, pH 8.5,was used at 32° C. The protease assays were typically performed induplicate. In most experiments to determine stability measurements, theprotein and detergent were diluted using the above-mentioned buffer by1:1000, although in some experiments dilutions of were also 1:500 or1:200, in order to obtain readings where the absorbance of the blankswas less than 0.5. The microtiter spectrophotometer used in theseexperiments was a SpectraMax250® (Molecular Devices) and all assays wereconducted in medium protein-binding 96-well plates (Corning).

The results for the standards protein samples (e.g., trypsin andpurified metalloprotease) obtained in these assays indicated that therewas a non-linear response (a linear scale may be adequate only in anarrow assay range). Hence, the curve was fitted to a quadratic functionwhere f=y₀+ax²+bx; f is fit to y (SigmaPlot® v. 9; SPSS, Inc.). Thus, ifa linear equation was used to quantitate the amount of protein,inaccurate data were obtained; the quadratic equation was found to berequired in order to obtain accurate results. It is noted that themanufacturer's (Pierce) kit insert indicates that the results may befitted with “x” being a log scale.

Example 6 Effect of pH and LAS on Neutral Metalloprotease Activity

The pH optimum of the activity for 0.36 mg/mL of formulated nprE wasalso determined. The buffers investigated in this study were 50 mMsodium acetate over the pH range 3.5-5.5 (pKa=4.76), 50 mM MES bufferover the pH range 5.5 to 7.0 (pKa=6.10), and 50 mM Tris-HCl buffer at pH8.4. The pH optimum for formulated nprE was determined to be between 5.5and 6.0.

The effect of the detergent component LAS on the activity of 0.36 mg/mlof formulated nprE was investigated by incubation with 0 to 1% (w/v)LAS. The results are shown in the graph provided at FIG. 2. As theseresults indicate, the protease is significantly inactivated by thedetergent component, thereby necessitating a means to stabilize theprotease against this deleterious effect.

In some experiments, the high density liquid detergent (HDL) compositiondesignated as “TIDE® 2005,” provided by Procter & Gamble was used. Assupplied, this detergent contained all necessary components, except forthe neutral metalloprotease of the present invention.

Storage Stability in Liquid Detergent Base as a Function of Time

The stability test was performed in a mini-storage manner. Theconditions to be varied and the various concentrations of calcium andzinc chloride salts to be added were assessed using a matrix designedusing the FusionPro™ (S-Matrix) software. The following table summarizesthe conditions tested to ascertain the long-term storage stability ofneutral metalloprotease from B. amyloliquefaciens.

TABLE 6 Long-Term Storage Test Conditions Condition [CaCl₂] (mM) [ZnCl₂](mM) 1 15 — 2 7.5 7.5 3 — 15 4 — — 5 12 3 6 — — 7 — 15 8 7.5 7.5 9 15 —10 15 15 11 12 3

The final volume of each tested condition was 1 mL. TIDE® 2005 was dosedwith 0.36 mg enzyme/mL. Formulated culture fluid and purifiedrecombinant metalloprotease were incubated in the TIDE® 2005 base at 32°C. over a period of approximately 4 weeks. The storage stability of themetalloprotease in detergent was compared to the stability of theneutral metalloprotease in 50 mM MES buffer, pH 5.8.

Prior to testing, the samples were diluted 5 in 1000 using assay buffer(50 mM borate buffer, pH 8.5). The residual activity was determined andcompared relative to the neutral metalloprotease in assay buffer. Allmeasurements were determined in duplicate. Each sample was tested inparallel with appropriate control blanks (i.e., the detergent, bufferand any necessary additives being tested). The samples were then assayedas described in the instructions provided with the QuantiCleave™Protease Assay Kit (Pierce).

The results of these stability tests conducted over a 3-4 week periodare shown in FIG. 22. In TIDE® 2005, the neutral metalloprotease in theabsence of ions (i.e., no added salt) rapidly lost all of itsproteolytic/hydrolytic activity against casein. Indeed it was determinedthat less than 20% of the activity remained after less than 1 hour ofincubation. In contrast, incubation of nprE in TIDE® 2005 containingzinc ions (up to and including 15 mM) stabilized the protease andprevented proteolysis over a 7-day period. Thus, the presence of zincions in this formulation functioned well in maintaining at least 60% ofthe protease activity. Likewise, a concentration of 7.5 mM zinc ionsresulted in a similar stabilization effect. This concentration of zincions is exceeding low and is contemplated to find use in a variety ofdetergent formulations. In these experiments, no added effect wasprovided by the inclusion of calcium ions. Furthermore, the addition ofcalcium ions in excess of 15 mM, and up to and including 25 mM, inducedprecipitation when added to TIDE® 2005 base. Although it is not intendedthat the present invention be limited to any particular mechanism, itwas contemplated that the absence of an effect of added calcium ions onprotease stabilization in these experiments was the result of thedetergent composition.

For thermolysin, which displays 55% amino acid sequence identity withneutral metalloprotease from B. amyloliquefaciens (sequence alignmentperformed using CLUSTAL W, v. 1.82), it has been clearly shown that zincions are essential for activity, whereas the calcium ions andengineering of the calcium binding sites have been shown to play astabilization role (See e.g., Mansfield., et al., J. Biol. Chem.,272:11152-11156 [1997]; and Van den Berg et al., Biotechnol. Appl.Biochem., 30:35-40 [1999]).

In alternative embodiments, other cations (e.g., Co²⁺, Mn²⁺ and Fe²⁺)find use in the present invention for the stabilization of neutralmetalloprotease from B. amyloliquefaciens. This is in contrast to priordata that has indicated that none of these ions resulted in 100%restoration of specific activity (Holmquist. and Vallee, J. Biol. Chem.,249:4601-4607 [1974]). It is contemplated that these ions will affectstability by preventing the unfolding and subsequent proteolyticdegradation of the metalloprotease. However, it is not intended that thepresent invention be limited to any particular mechanism of action.

Example 7 NprE Protease Production in B. subtilis Using the nprEExpression Vector pUBnprE

In this Example, experiments conducted to produce NprE protease in B.subtilis, in particular, the methods used in the transformation ofplasmid pUBnprE into B. subtilis are described. Transformation wasperformed as known in the art (See e.g., WO 02/14490, incorporatedherein by reference). The DNA sequence (nprE leader, nprE pro and nprEmature DNA sequence from B. amyloliquefaciens) provided below, encodesthe NprE precursor protein:

(SEQ ID NO: 12) GTGGGTTTAGGTAAGAAATTGTCTGTTGCTGTCGCCGCTTCCTTTATGAGTTTAACCATCAGTCTGCCGGGTGTTCAGGCCGCTGAGAATCCTCAGCTTAAAGAAAACCTGACGAATTTTGTACCGAAGCATTCTTTGGTGCAATCAGAATTGCCTTCTGTCAGTGACAAAGCTATCAAGCAATACTTGAAACAAAACGGCAAAGTCTTTAAAGGCAATCCTTCTGAAAGATTGAAGCTGATTGACCAAACGACCGATGATCTCGGCTACAAGCACTTCCGTTATGTGCCTGTCGTAAACGGTGTGCCTGTGAAAGACTCTCAAGTCATTATTCACGTCGATAAATCCAACAACGTCTATGCGATTAACGGTGAATTAAACAACGATGTTTCCGCCAAAACGGCAAACAGCAAAAAATTATCTGCAAATCAGGCGCTGGATCATGCTTATAAAGCGATCGGCAAATCACCTGAAGCCGTTTCTAACGGAACCGTTGCAAACAAAAACAAAGCCGAGCTGAAAGCAGCAGCCACAAAAGACGGCAAATACCGCCTCGCCTATGATGTAACCATCCGCTACATCGAACCGGAACCTGCAAACTGGGAAGTAACCGTTGATGCGGAAACAGGAAAAATCCTGAAAAAGCAAAA CAAAGTGGAGCATGCCGCCACAACCGGAACAGGTACGACTCTTAAAGGAAAAACGGTCTCATTAAATATTTCTTCTGAAAGCGGCAAATATGTGCTGCGCGATCTTTCTAAACCTACCGGAACACAAATTATTACGTACGATCTGCAAAACCGCGAGTATAACCTGCCGGGCACACTCGTATCCAGCACCACAAACCAGTTTACAACTTCTTCTCAGCGCGCTGCCGTTGATGCGCATTACAACCTCGGCAAAGTGTATGATTATTTCTATCAGAAGTTTAATCGCAACAGCTACGACAATAAAGGCGGCAAGATCGTATCCTCCGTTCATTACGGCAGCAGATACAATAACGCAGCCTGGATCGGCGACCAAATGATTTACGGTGACGGCGACGGTTCATTCTTCTCACCTCTTTCCGGTTCAATGGACGTAACCGCTCATGAAATGACACATGGCGTTACACAGGAAACAGCCAACCTGAACTACGAAAATCAGCCGGGCGCTTTAAACGAATCCTTCTCTGATGTATTCGGGTACTTCAACGATACTGAGGACTGGGATATCGGTGAAGATATTACGGTCAGCCAGCCGGCTCTCCGCAGCTTATCCAATCCGACAAAATACGGACAGCCTGATAATTTCAAAAATTACAAAAACCTTCCGAACACTGATGCCGGCGACTACGGCGGCGTGCATACAAACAGCGGAATCCCGAACAAAGCCGCTTACAATACGATTACAAAAATCGGCGTGAACAAAGCGGAGCAGATTTACTATCGTGCTCTGACGGTATACCTCACTCCGTCATCAACTTTTAAAGATGCAAAAGCCGCTTTGATTCAATCTGCGCGGGACCTTTACGGCTCTCAAGATGCTGCAAGCGTAGAAGCTGCCTGGAA TGCAGTCGGATTGTAA

In the above sequence, bold indicates the DNA that encodes the matureNprE protease, standard font indicates the leader sequence (nprEleader), and underlined indicates the pro sequences (nprE pro). Theamino acid sequence (NprE leader, NprE pro and NprE mature DNA sequence)(SEQ ID NO:13) provided below, encodes the NprE precursor protein. Inthis sequence, underlined indicates the pro sequence and bold indicatesthe mature NprE protease. SEQ ID NO:17 provides the NprE pro-sequenceseparately from the mature NprE sequence and SEQ ID NO:18 provides themature NprE sequence. This sequence was used as the basis for making thevariant libraries described herein.

(SEQ ID NO: 13) MGLGKKLSVAVAASFMSLTISLPGVQAAENPQLKENLTNFVPKHSLVQSELPSVSDKAIKQYLKQNGKVFKGNPSERLKLIDQTTDDLGYKHFRYVPVVNGVPVKDSQVIIHVDKSNNVYAINGELNNDVSAKTANSKKLSANQALDHAYKAIGKSPEAVSNGTVANKNKAELKAAATKDGKYRLAYDVTIRYIEPEPAN WEVTVDAETGKILKKQNKVEHAATTGTGTTLKGKTVSLNISSESGKYVLRDLSKPTGTQIITYDLQNREYNLPGTLVSSTTNQFTTSSQRAAVDAHYNLGKVYDYFYQKFNRNSYDNKGGKIVSSVHYGSRYNNAAWIGDQMIYGDGDGSFFSPLSGSMDVTAHEMTHGVTQETANLNYENQPGALNESFSDVFGYFNDTEDWDIGEDITVSQPALRSLSNPTKYGQPDNFKNYKNLPNTDAGDYGGVHTNSGIPNKAAYNTITKIGVNKAEQIYYRALTVYLTPSSTFKDAKAALIQSA RDLYGSQDAASVEAAWNAVGL(SEQ ID NO: 17) AENPQLKENLTNFVPKHSLVQSELPSVSDKAIKQYLKQNGKVFKGNPSERLKLIDQTTDDLGYKHFRYVPVVNGVPVKDSQVIIHVDKSNNVYAINGELNNDVSAKTANSKKLSANQALDHAYKAIGKSPEAVSNGTVANKNKAELKAAATKDGKYRLAYDVTIRYIEPEPANWEVTVDAETGKILKKQNKVEH (SEQ ID NO: 18)AATTGTGTTLKGKTVSLNISSESGKYVLRDLSKPTGTQIITYDLQNREYNLPGTLVSSTTNQFTTSSQRAAVDAHYNLGKVYDYFYQKFNRNSYDNKGGKIVSSVHYGSRYNNAAWIGDQMIYGDGDGSFFSPLSGSMDVTAHEMTHGVTQETANLNYENQPGALNESFSDVFGYFNDTEDWDIGEDITVSQPALRSLSNPTKYGQPDNFKNYKNLPNTDAGDYGGVHTNSGIPNKAAYNTITKIGVNKAEQIYYRALTVYLTPSSTFKDAKAALIQSARDLYGSQDAASVEAAWNAVGL

The pUBnprE expression vector was constructed by amplifying the nprEgene from the chromosomal DNA of B. amyloliquefaciens by PCR using twospecific primers:

Oligo AB1740: (SEQ ID NO: 19)CTGCAGGAATTCAGATCTTAACATTTTTCCCCTATCATTTTTCCCG Oligo AB1741:(SEQ ID NO: 20) GGATCCAAGCTTCCCGGGAAAAGACATATATGATCATGGTGAAGCC

PCR was performed on a thermocycler with Phusion High Fidelity DNApolymerase (Finnzymes. The PCR mixture contained 10 μl 5× buffer(Finnzymes Phusion), 1 μl 10 mM dNTP's, 1.5 μl DMSO, 1 μl of eachprimer, 1 μl Finnzymes Phusion DNA polymerase, 1 μl chromosomal DNAsolution 50 ng/μl, 34.5 μl MilliQ water. The following protocol wasused:

PCR protocol:

1) 30 sec 98° C.;

2) 10 sec 98° C.;

3) 20 sec 55° C.;

4) 1 min 72° C.;

5) 25 cycles of steps 2 to 4; and

6) 5 min 72° C.

This resulted in a 1.9 kb DNA fragment which was digested using BglIIand BclI DNA restriction enzymes. The multicopy Bacillus vector pUB110(See e.g., Gryczan, J. Bacteriol., 134:318-329 [1978]) was digested withBamHI. The PCR fragment×BglII×BclI was then ligated in the pUB110× BamHIvector to form pUBnprE expression vector (See, FIG. 14).

pUBnprE was transformed to a B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE,degUHy32, ΔamyE::(xylR,pxylA-comK) strain. Transformation into B.subtilis was performed as described in WO 02/14490, incorporated hereinby reference. Selective growth of B. subtilis transformants harboringthe pUBnprE vector was performed in shake flasks containing 25 ml MBDmedium (a MOPS based defined medium), with 20 mg/L neomycin. MBD mediumwas made essentially as known in the art (See, Neidhardt et al., J.Bacteriol., 119: 736-747 [1974]), except that NH₄Cl₂, FeSO₄, and CaCl₂were left out of the base medium, 3 mM K₂HPO₄ was used, and the basemedium was supplemented with 60 mM urea, 75 g/L glucose, and 1% soytone.Also, the micronutrients were made up as a 100× stock containing in oneliter, 400 mg FeSO₄.7H₂O, 100 mg MnSO₄.H₂O, 100 mg ZnSO₄.7H₂O, 50 mgCuCl₂.2H₂O, 100 mg CoCl₂.6H₂O, 100 mg NaMoO₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O,10 ml of 1M CaCl₂, and 10 ml of 0.5 M sodium citrate. The culture wasincubated for three days at 37° C. in an incubator/shaker (Infors). Thisculture resulted in the production of secreted NprE protease withproteolytic activity as demonstrated by protease assays. Gel analysiswas performed using NuPage Novex 10% Bis-Tris gels (Invitrogen, Cat. No.NP0301BOX). To prepare samples for analysis, 2 volumes of supernatantwere mixed with 1 volume 1M HCl, 1 volume 4'LDS sample buffer(Invitrogen, Cat. No. NP0007), and 1% PMSF (20 mg/ml) and subsequentlyheated for 10 minutes at 70° C. Then, 25 μL of each sample were loadedonto the gel, together with 10 μL of SeeBlue plus 2 pre-stained proteinstandards (Invitrogen, Cat. No. LC5925). The results clearlydemonstrated that the nprE cloning strategy described in this exampleyield active NprE produced by B. subtilis.

Example 8 Generation of nprE Site Evaluation Libraries (SELs)

In this Example, methods used in the construction of nprE SELs aredescribed. The pUBnprE vector, containing the nprE expression cassettedescribed above, served as template DNA. This vector contains a uniqueBglII restriction site, which was utilized in the site evaluationlibrary construction.

The pUBnprE expression vector, primers, synthesized at Invitrogen(desalted, 50 nmol scale) were used to generate the libraries. Thesequences of the primers are provided in Table 8-1.

To construct a nprE site evaluation library, three PCR reactions wereperformed, including two mutagenesis PCRs to introduce the mutated codonof interest in the mature nprE DNA sequence and a third PCR used to fusethe two mutagenesis PCRs in order to construct the pUBnprE expressionvector including the desired mutated codon in the mature nprE sequence.

The method of mutagenesis was based on the codon-specific mutationapproach, in which the creation of all possible mutations at a time in aspecific DNA triplet was performed using a forward and reverseoligonucleotide primer with a length of 25 to 45 nucleotides enclosing aspecific designed triple DNA sequence NNS ((A,C,T or G), (A,C,T or G),(C or G)) that corresponded with the sequence of the codon to be mutatedand guaranteed random incorporation of nucleotides at that specific nprEmature codon. The number listed in the primer names (See, Table 8-1)corresponds with the specific nprE mature codon position.

Two additional primers used to construct the site evaluation librariescontained the BglII restriction site together with a part of the pUBnprEDNA sequence flanking the BglII restriction site. These primers wereproduced by Invitrogen (50 nmole scale, desalted) and are listed inTable 8-1.

TABLE 8-1 Primer Sequences Primer Name Primer Sequence and SEQ ID NO:pUB-Bg1II-FW GTCAGTCAGATCTTCCTTCAGGTTATGACC (SEQ ID NO: 21) pUB-Bg1II-RVGTCTCGAAGATCTGATTGCTTAACTGCTTC (SEQ ID NO: 22)Specific nprE Forward Mutagenesis Primers nprE4FGTGGAGCATGCCGCCACANNSGGAACAGGTACGACTCTTAA (SEQ ID NO: 23) nprE12FCAGGTACGACTCTTAAANNSAAAACGGTCTCATTAAATAT (SEQ ID NO: 24) nprE13FGTACGACTCTTAAAGGANNSACGGTCTCATTAAATATTTC (SEQ ID NO: 25) nprE14FCGACTCTTAAAGGAAAANNSGTCTCATTAAATATTTC (SEQ ID NO: 26) nprE23FCATTAAATATTTCTTCTGAANNSGGCAAATATGTGCTGCG (SEQ ID NO: 27) nprE24FTAAATATTTCTTCTGAAAGCNNSAAATATGTGCTGCGCGATC (SEQ ID NO: 28) nprE33FGTGCTGCGCGATCTTTCTNNSCCTACCGGAACACAAATTAT (SEQ ID NO: 29) nprE45FAAATTATTACGTACGATCTGNNSAACCGCGAGTATAACCTG (SEQ ID NO: 30) nprE46FTTATTACGTACGATCTGCAANNSCGCGAGTATAACCTGCC (SEQ ID NO: 31) nprE47FCGTACGATCTGCAAAACNNSGAGTATAACCTGCCGGG (SEQ ID NO: 32) nprE49FGATCTGCAAAACCGCGAGNNSAACCTGCCGGGCACACTC (SEQ ID NO: 33) nprE50FCTGCAAAACCGCGAGTATNNSCTGCCGGGCACACTCGTATC (SEQ ID NO: 34) nprE54FGAGTATAACCTGCCGGGCNNSCTCGTATCCAGCACCAC (SEQ ID NO: 35) nprE58FCGGGCACACTCGTATCCNNSACCACAAACCAGTTTAC (SEQ ID NO: 36) nprE59FGCACACTCGTATCCAGCNNSACAAACCAGTTTACAAC (SEQ ID NO: 37) nprE60FCACTCGTATCCAGCACCNNSAACCAGTTTACAACTTC (SEQ ID NO: 38) nprE65FCCACAAACCAGTTTACANNSTCTTCTCAGCGCGCTGC (SEQ ID NO: 39) nprE66FCAAACCAGTTTACAACTNNSTCTCAGCGCGCTGCCGTTG (SEQ ID NO: 40) nprE87FGTGTATGATTATTTCTATNNSAAGTTTAATCGCAACAG (SEQ ID NO: 41) nprE90FATTATTTCTATCAGAAGTTTNNSCGCAACAGCTACGACAATAA (SEQ ID NO: 42) nprE96FTTAATCGCAACAGCTACGACNNSAAAGGCGGCAAGATCGTATC (SEQ ID NO: 43) nprE97FGCAACAGCTACGACAATNNSGGCGGCAAGATCGTATC (SEQ ID NO: 44) nprE100FCTACGACAATAAAGGCGGCNNSATCGTATCCTCCGTTCATTA (SEQ ID NO: 45) nprE186FGAGGACTGGGATATCGGTNNSGATATTACGGTCAGCCAG (SEQ ID NO: 46) nprE196FGTCAGCCAGCCGGCTCTCNNSAGCTTATCCAATCCGAC (SEQ ID NO: 47) nprE211FGACAGCCTGATAATTTCNNSAATTACAAAAACCTTCC (SEQ ID NO: 48) nprE214FGATAATTTCAAAAATTACNNSAACCTTCCGAACACTGATG (SEQ ID NO: 49) nprE228FGCGACTACGGCGGCGTGNNSACAAACAGCGGAATCCC (SEQ ID NO: 50) nprE280FCTTTGATTCAATCTGCGNNSGACCTTTACGGCTCTCAAG (SEQ ID NO: 51)Specific nprE Reverse Mutagenesis Primers nprE4RTTAAGAGTCGTACCTGTTCCSNNTGTGGCGGCATGCTCCAC (SEQ ID NO: 52) nprE12RATATTTAATGAGACCGTTTTSNNTTTAAGAGTCGTACCTG (SEQ ID NO: 53) nprE13RGAAATATTTAATGAGACCGTSNNTCCTTTAAGAGTCGTAC (SEQ ID NO: 54) nprE14RGAAATATTTAATGAGACSNNTTTTCCTTTAAGAGTCG (SEQ ID NO: 55) nprE23RCGCAGCACATATTTGCCSNNTTCAGAAGAAATATTTAATG (SEQ ID NO: 56) nprE24RGATCGCGCAGCACATATTTSNNGCTTTCAGAAGAAATATTTA (SEQ ID NO: 57) nprE33RATAATTTGTGTTCCGGTAGGSNNAGAAAGATCGCGCAGCAC (SEQ ID NO: 58) nprE45RCAGGTTATACTCGCGGTTSNNCAGATCGTACGTAATAATTT (SEQ ID NO: 59) nprE46RGGCAGGTTATACTCGCGSNNTTGCAGATCGTACGTAATAA (SEQ ID NO: 60) nprE47RCCCGGCAGGTTATACTCSNNGTTTTGCAGATCGTACG (SEQ ID NO: 61) nprE49RGAGTGTGCCCGGCAGGTTSNNCTCGCGGTTTTGCAGATC (SEQ ID NO: 62) nprE50RGATACGAGTGTGCCCGGCAGSNNATACTCGCGGTTTTGCAG (SEQ ID NO: 63) nprE54RGTGGTGCTGGATACGAGSNNGCCCGGCAGGTTATACTC (SEQ ID NO: 64) nprE58RGTAAACTGGTTTGTGGTSNNGGATACGAGTGTGCCCG (SEQ ID NO: 65) nprE59RGTTGTAAACTGGTTTGTSNNGCTGGATACGAGTGTGC (SEQ ID NO: 66) nprE60RGAAGTTGTAAACTGGTTSNNGGTGCTGGATACGAGTG (SEQ ID NO: 67) nprE65RGCAGCGCGCTGAGAAGASNNTGTAAACTGGTTTGTGG (SEQ ID NO: 68) nprE66RCAACGGCAGCGCGCTGAGASNNAGTTGTAAACTGGTTTG (SEQ ID NO: 69) nprE87RCTGTTGCGATTAAACTTSNNATAGAAATAATCATACAC (SEQ ID NO: 70) nprE90RTTATTGTCGTAGCTGTTGCGSNNAAACTTCTGATAGAAATAAT (SEQ ID NO: 71) nprE96RGATACGATCTTGCCGCCTTTSNNGTCGTAGCTGTTGCGATTAA (SEQ ID NO: 72) nprE97RGATACGATCTTGCCGCCSNNATTGTCGTAGCTGTTGC (SEQ ID NO: 73) nprE100RTAATGAACGGAGGATACGATSNNGCCGCCTTTATTGTCGTAG (SEQ ID NO: 74) nprE186RCTGGCTGACCGTAATATCSNNACCGATATCCCAGTCCTC (SEQ ID NO: 75) nprE196RGTCGGATTGGATAAGCTSNNGAGAGCCGGCTGGCTGAC (SEQ ID NO: 76) nprE211RGGAAGGTTTTTGTAATTSNNGAAATTATCAGGCTGTC (SEQ ID NO: 77) nprE214RCATCAGTGTTCGGAAGGTTSNNGTAATTTTTGAAATTATC (SEQ ID NO: 78) nprE228RGGGATTCCGCTGTTTGTSNNCACGCCGCCGTAGTCGC (SEQ ID NO: 79) nprE280RCTTGAGAGCCGTAAAGGTCSNNCGCAGATTGAATCAAAG (SEQ ID NO: 80)

Construction of each site evaluation library started with two primaryPCR amplifications using the pUB-BglII-FW primer and a specific nprEreverse mutagenesis primer. For the second PCR, the pUB-BglII-RV primerand a specific nprE forward mutagenesis primer (equal nprE mature codonpositions for the forward and reverse mutagenesis primers) were used.

The introduction of the mutations in the mature nprE sequence wasperformed using Phusion High-Fidelity DNA Polymerase (Finnzymes; Cat.no. F-530L). All PCRs were performed according to the Finnzymes protocolsupplied with the polymerase. The PCR conditions for the primary PCRswere:

For Primary PCR 1:

pUB-BglII-FW primer and a specific NPRE reverse mutagenesis primer—both1 μL (10 μM);

For Primary PCR 2:

pUB-BglII-RV primer and a specific NPRE forward mutagenesis primer—both1 μL (10 μM); together with

5 x Phusion HF buffer 10 μL 10 mM dNTP mixture 1 μL Phusion DNApolymerase 0.75 μL (2 units/μL) DMSO, 100% 1 μL pUBnprE template DNA 1μL (0.1-1 ng/μL) Distilled, autoclaved water up to 50 μL

The PCR program was: 30 seconds 98° C., 30× (10 seconds 98° C., 20seconds 55° C., 1.5 minute 72° C.) and 5 min 72° C., performed in aPTC-200 Peltier thermal cycle (MJ Research). The PCR experiments resultin two fragments of approximately 2 to 3 kB, which had about 30nucleotide base overlap around the NPRE mature codon of interest.Fragments were fused in a third PCR reaction using these twoaforementioned fragments and the forward and reverse BglII primers. Thefusion PCR reaction was carried out in the following solution:

pUB-BglII-FW primer and pUB-BglII-RV both 1 μL (10 μM) primer 5 xPhusion HF buffer 10 μL 10 mM dNTP mixture 1 μL Phusion DNA polymerase0.75 μL (2 units/μL) DMSO, 100% 1 μL primary PCR 1 reaction mix 1 μLprimary PCR 2 reaction mix 1 μL Distilled, autoclaved water up to 50 μL

The PCR fusion program was as follows: 30 seconds 98° C., 30× (10seconds 98° C., 20 seconds 55° C., 2:40 minute 72° C.) and 5 min 72° C.,in a PTC-200 Peltier thermal cycler (MJ Research).

The amplified linear 6.5 Kb fragment was purified using the Qiaquick PCRpurification kit (Qiagen, Cat. no. 28106) and digested with BglIIrestriction enzyme to create cohesive ends on both sides of the fusionfragment:

-   -   35 μL purified linear DNA fragment    -   4 μL REACT® 3 buffer (Invitrogen)    -   1 μL BglII, 10 units/ml (Invitrogen)        Reaction conditions: 1 hour, 30° C.

Ligation of the BglII digested and purified using Qiaquick PCRpurification kit (Qiagen, Cat. no. 28106) fragment results in circularand multimeric DNA containing the desired mutation:

-   -   30 μL of purified BglII digested DNA fragment    -   8 μL T4 DNA Ligase buffer (Invitrogen® Cat. no. 46300-018)    -   1 μL T4 DNA Ligase, 1 unit/μL (Invitrogen® Cat. no. 15224-017)        Reaction conditions: 16-20 hours, 16°

Subsequently, the ligation mixture was transformed to a B. subtilis(ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::(xylR,pxylA-comK) strain.Transformation to B. subtilis was performed as described in WO 02/14490,incorporated herein by reference. For each library, 96 single colonieswere picked and grown in MOPS media with neomycin and 1.25 g/L yeastextract for sequence analysis (BaseClear) and screening purposes. Eachlibrary included a maximum of 19 nprE site-specific variants.

The variants were produced by growing the B. subtilis SEL transformantsin 96 well MTP at 37° C. for 68 hours in MBD medium with 20 mg/Lneomycin and 1.25 g/L yeast extract (See, above).

Example 9 Generation of nprE Combinatorial Libraries (RCLs)

In this Example, methods used to generate nprE combinatorial librariesare described. For this enzyme, one property was chosen as the propertythat needed to be changed the most. This property is defined herein asthe “primary property.” All other properties were “secondary properties”for the purpose of combinatorial library design. The basic strategy forimproving a protein as used herein, was to combine mutations thatimprove the primary property and also maintain or improve the secondaryproperties. The site evaluation data were used to identify thosemutations which improved the primary property while maintaining orimproving the secondary properties. Mutations that were to be combinedwere identified by their Performance Index (PI or Pi) and associatedΔΔG_(app) values.

The “Apparent Free Energy Change” (ΔΔG_(app)) as used herein is definedas:ΔΔG _(app) =−RT Ln(P _(variant) /P _(parent))where P_(variant) is the performance value for the variant andP_(parent) is the performance value for the parent enzyme under the sameconditions. The ratio P_(variant)/P_(parent) is defined as theperformance index (Pi) for the property. The ΔΔG_(app) values wereexpected to behave in a similar fashion to actual ΔΔG values for datadistributions and additivity. However, since ΔΔG represents the maximumamount of work that can be carried out by the variant compared to theparent enzyme, the quantity ΔΔG_(app) generally underestimates the ΔΔGand may lead to results that appear synergistic in that the propertiesof two additive positions may be greater than the value predicted byadding their ΔΔG_(app) values together.

For example, when TIDE® stability is the primary property and BMIactivity is the secondary property, mutations that have ΔΔG_(app)values<0 (Pi>1) and BMI ΔΔG_(app) values<0.06 (Pi>0.9) may be chosen forcombination. Indeed, these relationships were explored in theseexperiments.

To produce the variants used in these experiments, synthetic nprElibrary fragments, containing multiple mutations at multiple nprE matureDNA positions, were produced by GeneArt (Geneart). These 1.5 kB nprElibrary fragments were digested with DNA restriction enzymes PvuI andAvaI, purified and ligated in the 5 kB pUB vector fragment (alsodigested with DNA restriction enzymes PvuI and AvaI) by a ligasereaction using T4 DNA Ligase (Invitrogen®Cat. no. 15224-017).

To transform the ligation reaction mix directly into Bacillus cells, thelibrary DNA (nprE library fragment mix ligated in pUB vector fragment)was amplified using the TempliPhi kit (Amersham cat. #25-6400). For thispurpose, 1 μL of the ligation reaction mix was mixed with 5 μL of samplebuffer from the TempliPhi kit and heated for 3 minutes at 95° C. todenature the DNA. The reaction was placed on ice to cool for 2 minutesand then spun down briefly. Next, 5 μL of reaction buffer and 0.2 μL ofphi29 polymerase from the TempliPhi kit were added, and the reactionswere incubated at 30° C. in an MJ Research PCR machine for 4 hours. Thephi29 enzyme was heat inactivated in the reactions by incubation at 65°C. for 10 min in the PCR machine.

For transformation of the libraries into Bacillus, 0.1 μL of theTempliPhi amplification reaction product was mixed with 500 μL ofcompetent B. subtilis cells (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32,ΔamyE::(xylR,pxylA-comK) followed by vigorous shaking at 37° C. for 1hour. Then, 100 and 500 μL were plated on HI-agar plates containing 20mg/L neomycin and 0.5% skim milk. In general, transformation to B.subtilis was performed as described in WO 02/14490, incorporated hereinby reference. B. subtilis nprE combinatorial libraries, constructed bythis method are contemplated to contain wild type amino acids at one ormore of the positions targeted for mutagenesis.

The variants obtained in these libraries were then tested for theirstability in TIDE® and their performance in BMI wash performance testsas described herein. Table 9 provides performance indices for thevariants tested in the BMI assay. In this Table, “Pos.” indicates theposition in the NprE amino acid sequence that was changed, and “AA”indicates the amino acid substitution made for each variant.

TABLE 9 Results (Performance Indices) for Tested Variants TIDE TIDE(−)TIDE(+) BMI BMI Pos. Variant AA (−) ΔΔG TIDE(+) ΔΔG Pi ΔΔG 4 T004L L0.80 0.13 1.13 −0.07 1.01 0.00 23 S023Y Y 1.08 −0.05 1.13 −0.07 1.02−0.01 23 S023W W 1.12 −0.07 1.13 −0.07 1.29 −0.15 23 S023N N 1.33 −0.171.10 −0.06 0.95 0.03 23 S023T T 0.88 0.07 1.06 −0.03 0.91 0.05 23 S023GG 1.29 −0.15 1.06 −0.03 0.92 0.05 23 S023R R 0.98 0.01 1.06 −0.03 1.46−0.22 23 S023L L 0.90 0.06 1.03 −0.02 1.24 −0.13 23 S023M M 1.04 −0.021.03 −0.02 1.09 −0.05 23 S023V V 0.82 0.12 1.02 −0.01 0.93 0.04 23 S023KK 1.01 −0.01 1.02 −0.01 1.50 −0.24 24 G024Y Y 0.60 0.30 1.11 −0.06 1.10−0.06 24 G024W W 0.36 0.60 1.10 −0.06 1.20 −0.11 24 G024M M 0.71 0.201.09 −0.05 1.12 −0.07 24 G024F F 0.50 0.41 1.08 −0.04 1.19 −0.10 24G024L L 0.49 0.42 1.07 −0.04 1.22 −0.12 24 G024H H 0.80 0.13 1.05 −0.031.17 −0.09 24 G024K K 0.55 0.35 1.04 −0.02 1.55 −0.26 24 G024T T 0.570.33 1.03 −0.02 0.94 0.04 24 G024R R 0.56 0.34 1.02 −0.01 1.47 −0.23 46N046Q Q 0.88 0.08 1.07 −0.04 1.22 −0.12 47 R047K K 1.12 −0.07 1.09 −0.051.15 −0.08 50 N050F F 1.07 −0.04 1.07 −0.04 1.38 −0.19 50 N050Y Y 1.000.00 1.04 −0.02 1.27 −0.14 50 N050W W 1.01 −0.01 1.04 −0.02 1.46 −0.2250 N050P P 1.23 −0.12 1.03 −0.02 1.12 −0.07 54 T054H H 1.08 −0.04 1.11−0.06 1.17 −0.09 54 T054K K 1.03 −0.02 1.11 −0.06 1.47 −0.23 54 T054L L1.09 −0.05 1.08 −0.05 1.26 −0.14 54 T054N N 0.97 0.02 1.07 −0.04 1.25−0.13 54 T054Y Y 1.14 −0.08 1.07 −0.04 1.08 −0.04 54 T054W W 1.02 −0.011.07 −0.04 1.22 −0.12 54 T054S S 0.99 0.01 1.05 −0.03 1.03 −0.02 54T054I I 1.09 −0.05 1.04 −0.02 1.34 −0.17 54 T054R R 0.96 0.02 1.04 −0.021.46 −0.22 54 T054Q Q 1.09 −0.05 1.03 −0.02 1.23 −0.12 54 T054F F 0.980.01 1.03 −0.02 1.16 −0.09 54 T054V V 1.14 −0.08 1.01 −0.01 1.11 −0.0659 T059R R 0.76 0.16 1.28 −0.14 1.56 −0.26 59 T059W W 0.56 0.34 1.26−0.14 1.32 −0.16 59 T059K K 0.99 0.00 1.16 −0.09 1.60 −0.28 59 T059N N0.98 0.01 1.15 −0.08 1.16 −0.09 59 T059G G 0.94 0.04 1.13 −0.07 1.11−0.06 59 T059P P 1.18 −0.10 1.12 −0.07 1.19 −0.10 59 T059M M 1.04 −0.021.10 −0.06 1.10 −0.05 59 T059H H 0.98 0.01 1.07 −0.04 1.32 −0.16 59T059S S 1.09 −0.05 1.04 −0.03 0.91 0.06 59 T059A A 1.05 −0.03 1.04 −0.020.96 0.03 59 T059Q Q 1.05 −0.03 1.04 −0.02 1.31 −0.16 59 T059I I 0.640.26 1.01 −0.01 1.43 −0.21 60 T060N N 0.79 0.14 1.03 −0.02 1.07 −0.04 66S066Q Q 0.75 0.17 1.01 −0.01 1.12 −0.07 66 S066N N 1.08 −0.05 1.01 −0.011.00 0.00 110 R110K K 1.08 −0.04 1.04 −0.02 1.05 −0.03 119 D119H H 1.03−0.02 1.15 −0.08 1.16 −0.09 129 S129I I 2.32 −0.49 1.68 −0.30 0.98 0.01129 S129V V 2.34 −0.50 1.55 −0.26 1.01 0.00 129 S129Q Q 1.86 −0.37 1.44−0.21 0.99 0.00 129 S129T T 1.59 −0.27 1.36 −0.18 1.04 −0.02 129 S129L L1.70 −0.31 1.35 −0.18 1.01 −0.01 129 S129H H 1.60 −0.28 1.30 −0.15 1.17−0.09 129 S129Y Y 1.28 −0.14 1.06 −0.04 1.25 −0.13 129 S129A A 1.13−0.07 1.06 −0.03 1.12 −0.07 129 S129K K 1.18 −0.10 1.05 −0.03 1.33 −0.17130 F130L L 1.29 −0.15 1.52 −0.25 0.91 0.05 130 F130I I 1.18 −0.10 1.14−0.08 1.03 −0.02 130 F130V V 1.05 −0.03 1.06 −0.03 0.99 0.00 130 F130K K0.99 0.00 1.04 −0.02 1.26 −0.14 138 M138L L 1.11 −0.06 1.43 −0.21 0.950.03 152 E152H H 1.53 −0.25 1.36 −0.18 1.15 −0.08 152 E152W W 1.32 −0.161.31 −0.16 1.06 −0.03 152 E152F F 1.32 −0.16 1.15 −0.08 1.09 −0.05 179T179P P 1.33 −0.17 1.50 −0.24 1.04 −0.03 190 V190I I 1.37 −0.18 1.68−0.30 1.16 −0.09 220 D220P P 2.24 −0.47 2.66 −0.57 1.05 −0.03 220 D220EE 2.23 −0.47 2.44 −0.52 1.05 −0.03 243 T243I I 1.13 −0.07 1.17 −0.091.06 −0.03 263 T263W W 1.37 −0.18 1.40 −0.20 0.92 0.05 263 T263H H 1.03−0.02 1.01 −0.01 1.05 −0.01 273 A273H H 1.10 −0.06 1.14 −0.08 0.98 0.01282 L282M M 1.03 −0.01 1.16 −0.09 1.01 −0.01 282 L282F F 0.91 0.05 1.06−0.04 1.09 −0.05 282 L282Y Y 0.83 0.11 1.04 −0.02 0.92 0.05 285 S285R R1.08 −0.04 1.38 −0.19 1.23 −0.12 285 S285P P 1.11 −0.06 1.30 −0.16 0.980.01 285 S285W W 1.08 −0.05 1.28 −0.14 0.95 0.03 285 S285Q Q 1.06 −0.031.10 −0.05 0.98 0.01 285 S285K K 0.89 0.07 1.00 0.00 1.20 −0.10 286Q286R R 0.95 0.03 1.18 −0.10 1.14 −0.08 286 Q286P P 0.98 0.01 1.15 −0.080.97 0.02 286 Q286K K 0.93 0.04 1.09 −0.05 1.22 −0.12

Example 10 Alternative Method Generate nprE Site Evaluation Libraries(SELs) Via QuikChange® Mutagenesis

In this Example, alternative methods to generate nprE SELs aredescribed. As in Example 8, above, the pUBnprE vector served as thetemplate DNA source for the generation of nprE SELs. The majordifference between the two methods is that this method requiresamplification of the entire vector using complementary site-directedmutagenic primers.

Materials:

Bacillus strain containing the pUBnprE vector

Qiagen Plasmid Midi Kit (Qiagen cat #12143)

Ready-Lyse Lysozyme (Epicentre cat #R1802M)

dam Methylase Kit (New England Biolabs cat #M0222L)

Zymoclean Gel DNA Recovery Kit (Zymo Research cat #D4001)

nprE site-directed mutagenic primers, 100 nmole scale, 5′Phosphorylated, PAGE purified (Integrated DNA Technologies, Inc.)

QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene cat #200514)

MJ Research PTC-200 Peltier Thermal Cycler (Bio-Rad Laboratories)

1.2% agarose E-gels (Invitrogen cat #G5018-01)

TempliPhi Amplification Kit (GE Healthcare cat #25-6400-10)

Competent B. subtilis cells (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32,ΔamyE::(xylR,pxylA-comK)

Methods:

To obtain the pUBnprE vector, a single colony of a Bacillus straincontaining the pUBnprE vector was used to inoculate a 5 ml LB+10 ppmneomycin tube. This was the starter culture used in these methods. Theculture was grown at 37° C., with shaking at 225 rpm for 6 hours. Then,100 ml of fresh LB+10 ppm neomycin were inoculated with 1 ml of thestarter culture. This culture was grown overnight at 37° C., withshaking at 225 rpm. Following this incubation, the cell pellet washarvested by sufficient centrifugation to provide a cell pellet. Thecell pellet was resuspended in 10 ml Buffer P1 (Qiagen Plasmid MidiKit). Then, 10 ul of Ready-Lyse Lysozyme was added to the resuspendedcell pellet and incubated at 37° C. for 30 min. Then, the Qiagen PlasmidMidi Kit protocol was continued (using 10 ml of Buffer P2 and P3 toaccount for the increased volume of cell culture). After isolation ofpUBnprE vector from Bacillus, the concentration of pUBnprE vector wasquantitated. The vector was then dam methylated using the dam MethylaseKit (New England Biolabs), using the methods set forth in the kitprotocols, to methylate approximately 2 ug of pUBnprE vector per tube.The Zymoclean Gel DNA recovery kit was used to purify and concentratethe dam-methylated pUBnprE vector. The dam-methylated pUBnprE vector wasquantitated and then diluted to a working concentration of 50 ng/ul.Complementary site-directed mutagenic primers (1 ul of each primer at 10uM) (See, Table 10-1), were used in a PCR reaction in the QuikChangeMulti Site-Directed Mutagenesis Kit (Stratagene), following themanufacturer's protocol (e.g., 1 ul dam methylated pUBnprE vector (50ng/ul), 1 ul nprE site-directed Forward mutagenic primer (10 uM), 1 ulnprE site-directed Forward mutagenic primer (10 uM), 2.5 ul 10×QuikChange Multi Reaction buffer, 1 ul dNTP Mix, 1 ul QuikChange Multienzyme blend (2.5 U/ul), and 17.5 ul distilled, autoclaved water, toprovide a 25 ul total reaction mix). The nprE site evaluation librarieswere amplified using the following conditions: 95° C., for 1 min.(1^(st) cycle only), followed by 95° C. for 1 min., 55° C. for 1 min,65° C. for 13½ min., and repeat cycling 23 times. The reaction productwas stored at 4° C. overnight. Then, the reaction mixture underwent DpnIdigest treatment (supplied with QuikChange Multi Site-DirectedMutagenesis Kit) to digest parental pUBnprE vector, using themanufacturer's protocol (i.e., 1.5 ul DpnI restriction enzyme was addedto each tube and incubated at 37° C. for 3 hours; 2 ul of DpnI-digestedPCR reaction was then analyzed on a 1.2% E-gel for each nprE SEL toensure PCR reaction worked and that parental template was degraded).TempliPhi rolling circle amplification was then used to generate largeamounts of DNA for increasing library size of each nprE SEL, using themanufacturer's protocol (i.e., 1 ul DpnI treated QuikChange MultiSite-Directed Mutagenesis PCR, 5 ul TempliPhi Sample Buffer, 5 ulTempliPhi Reaction Buffer, and 0.2 ul TempliPhi Enzume Mix, for an ˜11ul total reaction; incubated at 30° C. for 3 hours; the TempliPhireaction was diluted by adding 200 ul distilled, autoclaved water andbriefly vortexed. Then, 1.5 ul of diluted TempliPhi material wastransformed into competent B. subtilis cells, and nprE SELs wereselected for using LA+10 ppm Neomycin+1.6% skim milk plates. Table 10-1provides the names, sequences and SEQ ID NOS for the primers used inthese experiments. All of the primers were synthesized by Integrated DNATechnologies, on 100 nmole scale, 5′-phosphorylated, and PAGE purified.

TABLE 10-1 Primers PRIMER SEQUENCE nprE-T4FGTGGAGCATGCCGCCACANNSGGAACAGGTACGACTCTTAAAGG (SEQ ID NO: 81) nprE-G12FCCGGAACAGGTACGACTCTTAAANNSAAAACGGTCTCATTAAATATTTCTTCTGAAAGC(SEQ ID NO: 82) nprE-Q45FCGGAACACAAATTATTACGTACGATCTGNNSAACCGCGAGTATAACCTGCC (SEQ ID NO: 83)nprE-Y49F AAACCGCGAGNNSAACCTGCCGGGCACACTCGTATCC (SEQ ID NO: 84)nprE-N50F GTACGATCTGCAAAACCGCGAGTATNNSCTGCCGGGCACACTCGTATCCAG(SEQ ID NO: 85) nprE-T65FCCAGCACCACAAACCAGTTTACANNSTCTTCTCAGCGCGCTGCCGTTG (SEQ ID NO: 86)nprE-D119F GCAGATACAATAACGCAGCCTGGATCGGCNNSCAAATGATTTACGGTGACGGCGAC(SEQ ID NO: 87) nprE-G128FCCAAATGATTTACGGTGACGGCGACNNSTCATTCTTCTCACCTCTTTCCGGTTC (SEQ ID NO: 88)nprE-F130F GGTGACGGCGACGGTTCANNSTTCTCACCTCTTTCCGGTCC (SEQ ID NO: 89)nprE-Q151F CATGAAATGACACATGGCGTTACANNSGAAACAGCCAACCTGAACTAC(SEQ ID NO: 90) nprE-E152FCATGAAATGACACATGGCGTTACACAGNNSACAGCCAACCTGAACTACG (SEQ ID NO: 91)nprE-N155F CATGGCGTTACACAGGAAACAGCCNNSCTGAACTACGAAAATCAGCCG(SEQ ID NO: 92) nprE-T179FCTGATGTATTCGGGTACTTCAACGATNNSGAGGACTGGGATATCGGTG (SEQ ID NO: 93)nprE-Y204F GCAGCTTATCCAATCCGACAAAANNSGGACAGCCTGATAATTTCAAAAATTAC(SEQ ID NO: 94) nprE-G205FGCAGCTTATCCAATCCGACAAAATACNNSCAGCCTGATAATTTCAAAAATTACAAAAACC(SEQ ID NO: 95) nprE-Y224F GAACACTGATGCCGGCGACNNSGGCGGCGTGCATACAAAC(SEQ ID NO: 96) nprE-T243FGAACAAAGCCGCTTACAATACGATTNNSAAAATCGGCGTGAACAAAGCG (SEQ ID NO: 97)nprE-V260F GCAGATTTACTATCGTGCTCTGACGNNSTACCTCACTCCGTCATCAACTTTTAAAG(SEQ ID NO: 98) nprE-Y261FGATTTACTATCGTGCTCTGACGGTANNSCTCACTCCGTCATCAACTTTTAAAG (SEQ ID NO: 99)nprE-T263F GTGCTCTGACGGTATACCTCNNSCCGTCATCAACTTTTAAAGATGC(SEQ ID NO: 100) nprE-A273FCCGTCATCAACTTTTAAAGATGCAAAANNSGCTTTGATTCAATCTGCGCGG (SEQ ID NO: 101)nprE-L282F GATTCAATCTGCGCGGGACNNSTACGGCTCTCAAGATGCTGC (SEQ ID NO: 102)nprE-S285F CGCGGGACCTTTACGGCNNSCAAGATGCTGCAAGCGTAG (SEQ ID NO: 103)nprE-A289F CCTTTACGGCTCTCAAGATGCTNNSAGCGTAGAAGCTGCCTGGAATG(SEQ ID NO: 104) nprE-A293FCTCAAGATGCTGCAAGCGTAGAANNSGCCTGGAATGCAGTCGGATTG (SEQ ID NO: 105)nprE-N296F GCAAGCGTAGAAGCTGCCTGGNNSGCAGTCGGATTGTAAACAAGAAAAG(SEQ ID NO: 106) nprE-G299FGAAGCTGCCTGGAATGCAGTCNNSTTGTAAACAAGAAAAGAGACCGGAAATCC (SEQ ID NO: 107)nprE-T60F CACACTCGTATCCAGCACCNNSAACCAGTTTACAACTTCTTCTCAG(SEQ ID NO: 108) nprE-R110F CTCCGTTCATTACGGCAGCNNSTACAATAACGCAGCCTGGATC(SEQ ID NO: 109) nprE-D139FCTCACCTCTTTCCGGTTCAATGNNSGTAACCGCTCATGAAATGACAC (SEQ ID NO: 110)nprE-T4R CCTTTAAGAGTCGTACCTGTTCCSNNTGTGGCGGCATGCTCCAC (SEQ ID NO: 111)nprE-G12R GCTTTCAGAAGAAATATTTAATGAGACCGTTTTSNNTTTAAGAGTCGTACCTGTTCCGG(SEQ ID NO: 112) nprE-Q45RGGCAGGTTATACTCGCGGTTSNNCAGATCGTACGTAATAATTTGTGTTCCG (SEQ ID NO: 113)nprE-Y49R GGATACGAGTGTGCCCGGCAGGTTSNNCTCGCGGTTTTGCAGATCGTAC(SEQ ID NO: 114) nprE-N50RCTGGATACGAGTGTGCCCGGCAGSNNATACTCGCGGTTTTGCAGATCGTAC (SEQ ID NO: 115)nprE-T65R CAACGGCAGCGCGCTGAGAAGASNNTGTAAACTGGTTTGTGGTGCTGG(SEQ ID NO: 116) nprE-D119RGTCGCCGTCACCGTAAATCATTTGSNNGCCGATCCAGGCTGCGTTATTGTATCTGC(SEQ ID NO: 117) nprE-G128RGAACCGGAAAGAGGTGAGAAGAATGASNNGTCGCCGTCACCGTAAATCATTTGG (SEQ ID NO: 118)nprE-F130R GGACCGGAAAGAGGTGAGAASNNTGAACCGTCGCCGTCACC (SEQ ID NO: 119)nprE-Q151R GTAGTTCAGGTTGGCTGTTTCSNNTGTAACGCCATGTGTCATTTCATG(SEQ ID NO: 120) nprE-E152RCGTAGTTCAGGTTGGCTGTSNNCTGTGTAACGCCATGTGTCATTTCATG (SEQ ID NO: 121)nprE-N155R CGGCTGATTTTCGTAGTTCAGSNNGGCTGTTTCCTGTGTAACGCCATG(SEQ ID NO: 122) nprE-T179RCACCGATATCCCAGTCCTCSNNATCGTTGAAGTACCCGAATACATCAG (SEQ ID NO: 123)nprE-Y204R GTAATTTTTGAAATTATCAGGCTGTCCSNNTTTTGTCGGATTGGATAAGCTGC(SEQ ID NO: 124) nprE-G205RGGTTTTTGTAATTTTTGAAATTATCAGGCTGSNNGTATTTTGTCGGATTGGATAAGCTGC(SEQ ID NO: 125) nprE-Y224R GTTTGTATGCACGCCGCCSNNGTCGCCGGCATCAGTGTTC(SEQ ID NO: 126) nprE-T243RCGCTTTGTTCACGCCGATTTTSNNAATCGTATTGTAAGCGGCTTTGTTC (SEQ ID NO: 127)nprE-V260R CTTTAAAAGTTGATGACGGAGTGAGGTASNNCGTCAGAGCACGATAGTAAATCTGC(SEQ ID NO: 128) nprE-Y261RCTTTAAAAGTTGATGACGGAGTGAGSNNTACCGTCAGAGCACGATAGTAAATC (SEQ ID NO: 129)nprE-T263R GCATCTTTAAAAGTTGATGACGGSNNGAGGTATACCGTCAGAGCAC(SEQ ID NO: 130) nprE-A273RCCGCGCAGATTGAATCAAAGCSNNTTTTGCATCTTTAAAAGTTGATGACGG (SEQ ID NO: 131)nprE-L282R GCAGCATCTTGAGAGCCGTASNNGTCCCGCGCAGATTGAATC (SEQ ID NO: 132)nprE-S285R CTACGCTTGCAGCATCTTGSNNGCCGTAAAGGTCCCGCG (SEQ ID NO: 133)nprE-A289R CATTCCAGGCAGCTTCTACGCTSNNAGCATCTTGAGAGCCGTAAAGG(SEQ ID NO: 134) nprE-A293RCAATCCGACTGCATTCCAGGCSNNTTCTACGCTTGCAGCATCTTGAG (SEQ ID NO: 135)nprE-N296R CTTTTCTTGTTTACAATCCGACTGCSNNCCAGGCAGCTTCTACGCTTGC(SEQ ID NO: 136) nprE-G299RGGATTTCCGGTCTCTTTTCTTGTTTACAASNNGACTGCATTCCAGGCAGCTTC (SEQ ID NO: 137)nprE-T60R CTGAGAAGAAGTTGTAAACTGGTTSNNGGTGCTGGATACGAGTGTG(SEQ ID NO: 138) nprE-R110R GATCCAGGCTGCGTTATTGTASNNGCTGCCGTAATGAACGGAG(SEQ ID NO: 139) nprE-D139RGTGTCATTTCATGAGCGGTTACSNNCATTGAACCGGAAAGAGGTGAG (SEQ ID NO: 140)nprE-S13SF GCGACGGTTCATTCTTCTCACCTCTTNNSGGTTCAATGGACGTAACCGCTC(SEQ ID NO: 141) nprE-G136FGCGACGGTTCATTCTTCTCACCTCTTTCCNNSTCAATGGACGTAACCGCTCATG (SEQ ID NO: 142)nprE-S137F CTTCTCACCTCTTTCCGGTNNSATGGACGTAACCGCTCATG (SEQ ID NO: 143)nprE-V140F CCTCTTTCCGGTTCAATGGACNNSACCGCTCATGAAATGACAC (SEQ ID NO: 144)nprE-S197F CAGCCAGCCGGCTCTCCGCNNSTTATCCAATCCGACAAAATACGGACAG(SEQ ID NO: 145) nprE-L198FCAGCCAGCCGGCTCTCCGCAGCNNSTCCAATCCGACAAAATACGGACAG (SEQ ID NO: 146)nprE-S199F CAGCCAGCCGGCTCTCCGCAGCTTANNSAATCCGACAAAATACGGACAGCC(SEQ ID NO: 147) nprE-L216FCAGCCTGATAATTTCAAAAATTACAAAAACNNSCCGAACACTGATGCCGGCGAC (SEQ ID NO: 148)nprE-P217F CAGCCTGATAATTTCAAAAATTACAAAAACCTTNNSAACACTGATGCCGGCGAC(SEQ ID NO: 149) nprE-N218FCAGCCTGATAATTTCAAAAATTACAAAAACCTTCCGNNSACTGATGCCGGCGACTAC(SEQ ID NO: 150) nprE-T219FCAGCCTGATAATTTCAAAAATTACAAAAACCTTCCGAACNNSGATGCCGGCGACTACGG(SEQ ID NO: 151) nprE-D220FCAGCCTGATAATTTCAAAAATTACAAAAACCTTCCGAACACTNNSGCCGGCGACTACGGCGGCG(SEQ ID NO: 152) nprE-A221FCAGCCTGATAATTTCAAAAATTACAAAAACCTTCCGAACACTGATNNSGGCGACTACGGCGGCGTG(SEQ ID NO: 153) nprE-G222F CCTTCCGAACACTGATGCCNNSGACTACGGCGGCGTGCATAC(SEQ ID NO: 154) nprE-Q286F CGGGACCTTTACGGCTCTNNSGATGCTGCAAGCGTAGAAGCTG(SEQ ID NO: 155) nprE-A297FGCGTAGAAGCTGCCTGGAATNNSGTCGGATTGTAAACAAGAAAAGAGACCGG (SEQ ID NO: 156)nprE-S135R GAGCGGTTACGTCCATTGAACCSNNAAGAGGTGAGAAGAATGAACCGTCGC(SEQ ID NO: 157) nprE-G136RCATGAGCGGTTACGTCCATTGASNNGGAAAGAGGTGAGAAGAATGAACCGTCGC (SEQ ID NO: 158)nprE-S137R CATGAGCGGTTACGTCCATSNNACCGGAAAGAGGTGAGAAG (SEQ ID NO: 159)nprE-V140R GTGTCATTTCATGAGCGGTSNNGTCCATTGAACCGGAAAGAGG (SEQ ID NO: 160)nprE-S197R CTGTCCGTATTTTGTCGGATTGGATAASNNGCGGAGAGCCGGCTGGCTG(SEQ ID NO: 161) nprE-L198RCTGTCCGTATTTTGTCGGATTGGASNNGCTGCGGAGAGCCGGCTGGCTG (SEQ ID NO: 162)nprE-S199R GGCTGTCCGTATTTTGTCGGATTSNNTAAGCTGCGGAGAGCCGGCTGGCTG(SEQ ID NO: 163) nprE-L216RGTCGCCGGCATCAGTGTTCGGSNNGTTTTTGTAATTTTTGAAATTATCAGGCTG (SEQ ID NO: 164)nprE-P217R GTCGCCGGCATCAGTGTTSNNAAGGTTTTTGTAATTTTTGAAATTATCAGGCTG(SEQ ID NO: 165) nprE-N218RGTAGTCGCCGGCATCAGTSNNCGGAAGGTTTTTGTAATTTTTGAAATTATCAGGCTG(SEQ ID NO: 166) nprE-T219RCCGTAGTCGCCGGCATCSNNGTTCGGAAGGTTTTTGTAATTTTTGAAATTATCAGGCTG(SEQ ID NO: 167) nprE-D220RCGCCGCCGTAGTCGCCGGCSNNAGTGTTCGGAAGGTTTTTGTAATTTTTGAAATTATCAGGCTG(SEQ ID NO: 168) nprE-A221RCACGCCGCCGTAGTCGCCSNNATCAGTGTTCGGAAGGTTTTTGTAATTTTTGAAATTATCAGGCTG(SEQ ID NO: 169) nprE-G222R GTATGCACGCCGCCGTAGTCSNNGGCATCAGTGTTCGGAAGG(SEQ ID NO: 170) nprE-Q286R CAGCTTCTACGCTTGCAGCATCSNNAGAGCCGTAAAGGTCCCG(SEQ ID NO: 171) nprE-A297RCCGGTCTCTTTTCTTGTTTACAATCCGACSNNATTCCAGGCAGCTTCTACGC (SEQ ID NO: 172)

Example 11 Identification of nprE Homologues

In this Example, experiments conducted to identify npr homologues aredescribed. In particular, in this Example, experiments were conducted toclone neutral protease (npr) homologs from different and closely relatedBacillus species. The different species were chosen in order to explorethe diversity and properties from which these different species areisolated.

The various npr homologs explored included:

B. caldolyticus npr (P23384)

B. cereus nprC (P05806)

B. cereus E33L npr (AAY60523)

B. stearothermophilus nprT

B. subtilis nprB

B. subtilis nprE

B. thuringiensis nprB (AAK00602)

S. aureus aur (P81177)

FIG. 3 provides a sequence alignment of these homologs (SEQ IDNOS:173-181) and FIG. 4 (SEQ ID NOS:182-191) provides another sequencealignment of various other homologs.

In these experiments, the materials included:

Chromosomal DNA of B. subtilis strain I168

The following DNA plasmids were synthesized at DNA2.0 with B. subtiliscodon optimization:

pJ4:G01905 (B. thuringiensis nprB) (See, FIG. 6)

pJ4:G01906 (B. cereus E33L npr) (See, FIG. 7)

pJ4:G01907 (B. cereus nprC) (See, FIG. 8)

pJ4:G01908 (B. caldolyticus npr) (See, FIG. 9)

pJ4:G01909 (S. aureus aur) (See, FIG. 10)

pJ4:G01938 (S. stearothermophilus nprT) (See, FIG. 11)

pJHT vector (See, FIG. 12)

pAC vector (See, FIG. 13)

MJ Research PTC-200 Peltier Thermal Cycler (Bio-Rad Laboratories)

Primers (Operon Inc)

PfuUltra II Fusion HS DNA Polymerase (Stratagene)

Restriction endonucleases (Roche)

TOP10 chemically competent E. coli cells (Invitrogen)

B. subtilis competent cells ((ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32,ΔamyE::(xylR,pxylA-comK)

TABLE 11 Primers Primer Name Primer Sequence and SEQ ID NO: EL-689CGTCTTCAACAATTGTCCATTTTCTTCTGC (SEQ ID NO: 196) EL-693CAGACAATTTCTTACCTAAACCCACTCTTTACCCTCTCCTTTTA AAAAAATTC (SEQ ID NO: 197)EL-694 GAATTTTTTTAAAAGGAGAGGGTAAAGAGTGGGTTTAGGTAAGA AATTGTCTG(SEQ ID NO: 198) EL-695 GCTTATGGATCCCGTCGTTTCAGCTGAGAGAG(SEQ ID NO: 199) EL-696 GATGTCTTGGTCAAGTTGCGCACTCTTTACCCTCTCCTTTTAAAAAAATTC (SEQ ID NO: 200) EL-697GAATTTTTTTAAAAGGAGAGGGTAAAGAGTGCGCAACTTGACCA AGACATC (SEQ ID NO: 201)EL-698 GGCCGGTTTTTTATGTAAGCTTATAGAATGCCGACAGCCTCATA CG (SEQ ID NO: 202)EL-699 CGTATGAGGCTGTCGGCATTCTATAAGCTTACATAAAAAACCGG CCTTGG(SEQ ID NO: 203) EL-700 AATGGTGCATGCAAGGAGATGGCG (SEQ ID NO: 204) EL-755CGTCTTCAAGAATTCCTCCATTTTCTTCTGC (SEQ ID NO: 205) EL-733GCACCCAACATTGCACGTTTATTCACTCTTTACCCTCTCCTTTT AAAAAAATTC (SEQ ID NO: 206)EL-734 GAATTTTTTTAAAAGGAGAGGGTAAAGAGTGAATAAACGTGCAA TGTTGGGTGC(SEQ ID NO: 207) EL-735 GCTTATAAGCTTAATATACTCCAACCGCGTTG(SEQ ID NO: 208) EL-739 CCAGCATAGCGCGTTTGTTCACTCTTTACCCTCTCCTTTTAAAAAAATTC (SEQ ID NO: 209) EL-740GAATTTTTTTAAAAGGAGAGGGTAAAGAGTGAACAAACGCGCTA TGCTGG (SEQ ID NO: 210)EL-741 GCTTATAAGCTTAATAGACACCCACGGCATTAAACGCC (SEQ ID NO: 211) EL-742CAGGACAAGAGCTAAGGACTTTTTTTTCACTCTTTACCCTCTCC TTTTAAAAAAATTC(SEQ ID NO: 212) EL-743 GAATTTTTTTAAAAGGAGAGGGTAAAGAGTGAAAAAAAAGTCCTTAGCTCTTGTCCTG (SEQ ID NO: 213) EL-744 GCTTATAAGCTTAATTAATGCCGACGGCAC(SEQ ID NO: 214)A. Cloning of B. subtilis nprE

To construct the B. subtilis nprE plasmid, the amplified aprE promoterfragment (from pJHT vector) and B. subtilis nprE gene with terminatorfragment (from B. subtilis strain I168) were separately prepared. FIG.15 provides a schematic, illustrating the amplification of theindividual DNA fragments.

PCR Splice Overlap Extension (SOE) reaction was used to join the 2separate DNA fragments together. In this reaction, the followingreagents were combined: 1 ul aprE promoter DNA fragment, 1 ul B.subtilis nprE gene+Terminator fragment, 1 ul Primer EL-689 (25 uM), 1 ulPrimer EL-695 (25 uM), 5 ul 10× PfuUltra II Fusion HS DNA polymerasebuffer, 1 ul dNTP (10 mM), 1 ul PfuUltra II Fusion HS DNA polymerase,and 39 ul distilled, autoclaved water to provide a total reaction volumeof 50 ul. The PCR cycles were: 95° C. for 2 minutes (1^(st) cycle only),followed by 28 cycles of 95° C. for 30 seconds, 54° C. for 30 seconds,and 72° C. for 0:45 seconds.

The PCR fusion fragment of aprE promoter-B. subtilis nprEgene+Terminator was digested with MfeI and BamHI restrictionendonucleases. The pJHT vector was digested with EcoRI and BamHIrestriction endonucleases. The restriction endonuclease digested aprEpromoter-B. subtilis nprE gene+Terminator DNA fragment was then ligatedwith the restriction endonuclease digested pJHT vector. The ligationmixture was then transformed into TOP10 chemically competent E. colicells for selection on LA+50 ppm carbenicillin. After identification ofplasmids containing the correct DNA construct sequence for plasmidpEL501 (See, FIG. 16), transformed into competent B. subtilis cells forintegration into aprE promoter locus. Transformants were selected forprotease activity (i.e. skim milk clearing) on LA+5 ppmchloramphenicol+1.6% skim milk plates. Amplified strains were thentransferred to LA+25 ppm chloramphenicol+1.6% skim milk plates. Strainswere then transferred to LA+25 ppm chloramphenicol+1.6% skim milk platesfor amplification.

B. Cloning of B. subtilis nprB

To construct the B. subtilis nprB plasmid, amplified the aprE promoterfragment (from pJHT vector), B. subtilis nprB gene fragment (from B.subtilis strain I168), and Terminator fragment (from pJHT vector) wereseparately prepared. FIG. 17 provides a schematic diagram of theamplification of the individual DNA fragments.

PCR Splice Overlap Extension (SOE) reaction was used to join the threeseparate DNA fragments together. In this reaction, the followingreagents were combined: 1 ul aprE promoter DNA fragment, 1 ul B.subtilis nprB gene+Terminator fragment, 1 ul Terminator DNA fragment, 1ul Primer EL-689 (25 uM), 1 ul Primer EL-700 (25 uM), 5 ul 10× PfuUltraII Fusion HS DNA polymerase buffer, 1 ul dNTP (10 mM), 1 ul PfuUltra IIFusion HS DNA Polymerase, and 38 ul distilled, autoclaved water, for a50 ul total reaction volume. The PCR cycles were: 95° C. for 2 minutes(1^(st) cycle only), followed by 28 cycles of 95° C. for 30 seconds, 54°C. for 30 seconds, and 72° C. for 0:45 seconds.

The PCR fusion fragment of aprE promoter-B. subtilis nprBgene+Terminator was digested with MfeI and SphI restrictionendonucleases. The pJHT vector was digested with EcoRI and SphIrestriction endonucleases. The restriction endonuclease digested aprEpromoter-B. subtilis nprB gene+Terminator DNA fragment was then ligatedwith the restriction endonuclease digested pJHT vector. The ligationmixture was then transformed into TOP10 chemically competent E. colicells for selection on LA+50 ppm carbenicillin. After identification ofplasmids containing the correct DNA construct sequence for plasmidpEL508 (See, FIG. 18), transformed into competent B. subtilis cells forintegration into aprE promoter locus. Transformants were selected forprotease activity (i.e. skim milk clearing) on LA+5 ppmchloramphenicol+1.6% skim milk plates. Amplified strains were thentransferred to LA+25 ppm chloramphenicol+1.6% skim milk plates. Strainswere then transferred to LA+25 ppm chloramphenicol+1.6% skim milk platesfor amplification.

C. Cloning of B. stearothermophilus nprT

To construct the B. stearothermophilus nprT plasmid, the amplified aprEpromoter fragment (from pJHT vector) and B. stearothermophilus nprTfragment (from plasmid pJ4:G01938) were separately prepared. FIG. 19provides a schematic diagram of the amplification of the individual DNAfragments.

PCR Splice Overlap Extension (SOE) reaction was used to join the 2separate DNA fragments together. In this reaction, the followingreagents were combined: 1 ul aprE promoter DNA fragment, 1 ul B.stearothermophilus nprT gene fragment, 1 ul Primer EL-755 (25 uM), 1 ulPrimer EL-735 (25 uM), 5 ul 10× PfuUltra II Fusion HS DNA Polymerasebuffer, 1 ul dNTP (10 mM), 1 ul PfuUltra II Fusion HS DNA Polymerase,and 39 ul distilled, autoclaved water, to provide a total reactionvolume of 50 ul. The PCR cycles were: 95° C. for 2 minutes (1^(st) cycleonly), followed by 28 cycles of 95° C. for 30 seconds, 54° C. for 30seconds, and 72° C. for 0:45 seconds.

The PCR fusion fragment of aprE promoter-B. stearothermophilus nprTgene+Terminator was digested with EcoRI and HindIII restrictionendonucleases. The pAC vector was digested with EcoRI and HindIIIrestriction endonucleases. The restriction endonuclease digested aprEpromoter-B. stearothermophilus nprT DNA fragment was then ligated withthe restriction endonuclease digested pAC vector. TempliPhi rollingcircle amplification was then used to generate large amounts of theligated aprE promoter-B. stearothermophilus nprT pAC DNA molecule, usingthe manufacturer's protocol (i.e., 1 ul aprE promoter-B.stearothermophilus nprT pAC ligation reaction, 5 ul TempliPhi SampleBuffer, 5 ul TempliPhi Reaction Buffer, and 0.2 ul TempliPhi Enzume Mix,for an ˜11 ul total reaction; incubated at 30° C. for 3 hours). TheTempliPhi reaction was then transformed directly into competent B.subtilis cells for integration into aprE promoter locus, therebygenerating Bacillus strain EL560, confirmed by DNA sequencing analysis(See, FIG. 20). Transformants were selected for protease activity (i.e.skim milk clearing) on LA+5 ppm chloramphenicol+1.6% skim milk plates.Strains were then transferred to LA+25 ppm chloramphenicol+1.6% skimmilk plates for amplification.

D. Cloning of B. caldolyticus npr

To construct the B. caldolyticus npr plasmid, the amplified aprEpromoter fragment (from pJHT vector) and B. caldolyticus npr fragment(from plasmid pJ4:G01908) were separately prepared. FIG. 21 provides aschematic diagram of the amplification of the individual DNA fragments.

PCR Splice Overlap Extension (SOE) reaction was used to join the 2separate DNA fragments together. In this reaction, the followingreagents were combined: 1 ul aprE promoter DNA fragment, 1 ul B.caldolyticus npr gene fragment, 1 ul Primer EL-755 (25 uM), 1 ul PrimerEL-741 (25 uM), 5 ul 10× PfuUltra II Fusion HS DNA Polymerase buffer, 1ul dNTP (10 mM), 1 ul PfuUltra II Fusion HS DNA Polymerase, and 39 uldistilled, autoclaved water, to provide a total reaction volume of 50ul. The PCR cycles were: 95° C. for 2 minutes (1^(st) cycle only),followed by 28 cycles of 95° C. for 30 seconds, 54° C. for 30 seconds,and 72° C. for 0:45 seconds.

The PCR fusion fragment of aprE promoter-B. caldolyticus nprgene+Terminator was digested with EcoRI and HindIII restrictionendonucleases. The pAC vector was digested with EcoRI and HindIIIrestriction endonucleases. The restriction endonuclease digested aprEpromoter-B. caldolyticus npr DNA fragment was then ligated with therestriction endonuclease digested pAC vector. TempliPhi rolling circleamplification was then used to generate large amounts of the ligatedaprE promoter-B. caldolyticus npr pAC DNA molecule, using themanufacturer's protocol (i.e., 1 ul aprE promoter-B. caldolyticus nprpAC ligation reaction, 5 ul TempliPhi Sample Buffer, 5 ul TempliPhiReaction Buffer, and 0.2 ul TempliPhi Enzume Mix, for an ˜11 ul totalreaction; incubated at 30° C. for 3 hours). The TempliPhi reaction wasthen transformed directly into competent B. subtilis cells forintegration into aprE promoter locus, thereby generating Bacillus strainEL561 (See, FIG. 22), confirmed by DNA sequencing analysis.Transformants were selected for protease activity (i.e. skim milkclearing) on LA+5 ppm chloramphenicol+1.6% skim milk plates. Strainswere then transferred to LA+25 ppm chloramphenicol+1.6% skim milk platesfor amplification.

E. Cloning of B. thuringiensis nprB

To construct the B. thuringiensis nprB plasmid, the amplified aprEpromoter fragment (from pJHT vector) and B. thuringiensis nprB fragment(from plasmid pJ4:G01905) were separately prepared. FIG. 23 provides aschematic showing the amplification of the individual DNA fragments.

PCR Splice Overlap Extension (SOE) reaction was used to join the 2separate DNA fragments together. In this reaction, the followingreagents were combined: 1 ul aprE promoter DNA fragment, 1 ul B.thuringiensis nprB gene fragment, 1 ul Primer EL-755 (25 uM), 1 ulPrimer EL-744 (25 uM), 5 ul 10× PfuUltra II Fusion HS DNA Polymerasebuffer, 1 ul dNTP (10 mM), 1 ul PfuUltra II Fusion HS DNA Polymerase,and 39 ul distilled, autoclaved water, to provide a total reactionvolume of 50 ul. The PCR cycles were: 95° C. for 2 minutes (1^(st) cycleonly), followed by 28 cycles of 95° C. for 30 seconds, 54° C. for 30seconds, and 72° C. for 0:45 seconds.

The PCR fusion fragment of aprE promoter-B. thuringiensis nprBgene+Terminator was digested with EcoRI and HindIII restrictionendonucleases. The pAC vector was digested with EcoRI and HindIIIrestriction endonucleases. The restriction endonuclease digested aprEpromoter-B. thuringiensis nprB DNA fragment was then ligated with therestriction endonuclease digested pAC vector. The ligation mixture wasthen transformed into TOP10 chemically competent E. coli cells. Afteridentification of plasmids containing the correct DNA construct sequencefor plasmid pEL568, transformed into competent B. subtilis cells.Transformants were then selected for protease activity (i.e. skim milkclearing) on LA+5 ppm chloramphenicol+1.6% skim milk plates. DNA plasmidpreparation shows that plasmid pEL568 is stable in B. subtilis and doesnot integrate into the aprE promoter locus. FIG. 24 provides a map ofplasmid pEL568.

E. Homology Modeling

In yet additional embodiments, a homology model of the mature domain ofNprE from Bacillus amyloliquefaciens is provided. In these experiments,the protein sequence of the mature domain of the NprE sequence (SEQ IDNO:192) was run through the program BlastP (NCBI). This program matchesthe sequence to other known sequences of varying sequence identity. Fromthe output, sequences for which an X-ray crystal structures are knownwere identified. These sequences included S. aureus Npr (pdb id 1BQB) P.aeruginosa Npr (pdb id1EZM), B. thermolyticus thermolysin (pdb id 1KEI)and B. cereus Npr (pdb id 1NPC). FIG. 5 provides a sequence alignment ofthe various sequences analyzed. (SEQ ID NOS:192-195)

The sequence of the B. cereus Npr was found to be the most identical toNprE. B. thermolyticus thermolysin (pdb id 1 KEI) was excluded fromsubsequent steps as it is very similar to B. cereus Npr. A homologymodel was then prepared as detailed below, and all calculation were madeusing the program MOE (Chemical Computing).

First, the S. aureus Npr (pdb id 1BQB; SEQ ID NO:193) P. aeruginosa Npr(pdb id1EZM) (SEQ ID NO:194), and B. cereus Npr (pdb id 1NPC) (SEQ IDNO:195) sequences were aligned using known structure, in order to obtainthe most accurate sequence alignment (i.e., a structure based sequencealignment was produced). Next, the NprE mature sequence was aligned tothis structure based sequence alignment. Then, an initial homology modelof NprE was produced using the B. cereus Npr (pdb id 1NPC) structure asa template, and using the sequence alignment of NprE to B. cereus Nprproduced above. It was clear from inspection of this alignment thatwhereas B. cereus Npr contains four Ca²⁺ ion binding sites, while NprEonly contains two Ca²⁺ ion binding sites.

Finally, after the initial homology model was built, the metal ions(i.e., Zn²⁺, and two Ca²⁺) were computationally fixed, as were theirrespective protein ligands. The rest of the model structure wascomputationally minimized using the CHARMM22 parameter set, resulting inthe final homology model.

Example 12 Wash Performance Tests

In this Example, experiments conducted to determine the wash performanceof the metalloprotease of the present invention are described. All washperformance tests were performed under American wash conditions, asindicated below:

Laundry Wash Performance Tests Equipment: Terg-O-Tometer (US Testing) 6pot bench top model Temperature: 15° C./60° F. Wash Time: 12 minutesAgitation: 100 rpm Rinse Time: 3 minutes Water Hardness: 6 grains pergallon/105 ppm as CaCO₃ (3/1 Ca⁺²/Mg⁺²) Sud concentration: 1.6 g/lTIDE ® 2005 liquid detergent base Enzyme dosage: 0.00-0.55-2.75-5.50 mgactive protein/l wash solution Swatches: EMPA 116 Fixed, fixated at 20°C.: Blood, milk, ink on cotton (10 × 7.5 cm) EMPA 116 Unfixed: Blood,milk, ink on cotton (10 × 7.5 cm) Equest grass: Grass Medium scrubbed oncotton (10 × 7.5 cm) CFT C-10: Pigment, oil, milk on cotton (10 × 7.5cm) EMPA 221: Unsoiled cotton used as ballast (10 × 10 cm) 6 EMPA 116fixed + 2 EMPA 221 were put in one vessel 6 EMPA 116 unfixed + 2 EMPA221 were put in one vessel 6 Equest grass + 2 EMPA 221 were put in onevessel 6 CFT C-10 + 2 EMPA 221 were put in one vessel Drying conditions:Spin-drier, Grass stains were dried to the air, covered with darkclothes. The other stains were ironed. Measuring swatches: TristimulusMinolta Meter CR-300 with equation L (L * a * b), D65 Std. Illuminate,on a white background. Expressed on Delta % Soil Removal. 3 readings perswatch (before and after washing)% Stain Removal=(L value after washing−L value before washing)/(L_(0 white cotton) −L value before washing)×100%

All experiments were done in quadruplicate

The proteases were tested in a specially developed washing test, usingthree different cotton swatches, soiled with:

(a) milk, blood and ink (10.0×7.5 cm; EMPA), designated with the numbers116 unfixed and fixed (the stains were fixed at 20° C.);

(b) grass medium (10.0×7.5 cm; Equest); and

(c) pigment, oil and milk (10.0×7.5 cm designated with the numbers C-10CFT).

These experiments are described in greater detail below. The washingtests were performed in a bench top model Terg-O-Tometer (US Testing),equipped with six stainless steel test vessels. The stainless steel testvessels each contained 1.6 g of TIDE® 2005 liquid detergent base,dissolved in 1000 ml water of 105 ppm/6 grains per gallon, and were eachloaded with six of the same soiled cotton swatches and two extra ballastcotton swatches (EMPA 221). A selected protease (e.g., neutralmetalloprotease or another protease) was added to each vessel in aconcentration from 0.00 to 5.50 mg active protein per liter suds.

The tests were carried out for 12 minutes at 15° C./60° F., with anagitation of 100 rpm. After washing, the swatches were rinsed for 3minutes under cold tap water and placed in a spin-drier. The grassswatches were air-dried and covered with dark clothing to limit thesensitivity of the grass stains to light. All other swatches wereironed. All experiments were performed in quadruplicate.

The reflectance of the tested swatches was measured with a TristimulusMinolta Meter CR-300 using the equation L (L*a*b). Wash performancevalues were calculated using the following relationship:% Stain Removal=(L value after washing−L value before washing)/(L_(0 white cotton) −L value before washing)×100%

The results of the Terg-O-Tometer (TOM) assay for purified MULTIFECT®are shown in FIG. 26, and compared with those of subtilisin (BPN′Y217L), a serine alkaline protease. The TOM provided a fully operationaland valid means for discriminating between the different washperformances of various proteases (e.g., serine proteases, neutralmetalloprotease, and variants thereof). The TOM tests were performed onBMI and Equest medium-soiled grass surface with TIDE® 2005 as the basedetergent.

As indicated in FIG. 26, it was apparent that the purified neutralmetalloprotease clearly performed better in the wash test than theserine protease (BPN′ Y217L). In particular, 2.75 ppm of purifiedneutral metalloprotease was required to show a delta soil removal of˜10% compared to only 0.55 ppm of the serine protease (BPN′ Y217L) onthe Equest grass stain. The wash performance of the neutralmetalloprotease was also tested at low temperature and found to performvery well medium solid Equest stain fabric. Similar results wereobtained with purified commercially available MULTIFECT® neutralprotease as with the recombinant nprE produced as described above.

Example 13 Performance of nprE Variants in BMI-TIDE® 2× PerformanceAssay

In this Example, experiments conducted to assess the performance ofvarious nprE variants in the BMI assay outlined above are described. Themethods provided prior to Example 1 were used (See, “Microswatch Assayfor Testing Protease Performance”). The results for multiply-substitutedvariants with Performance Indices greater than one (PI>1) and those withPerformance Indices less than one (PI<1) are provided in the Tablesbelow.

In Table 13.1, data obtained for selected single-substitution variantsin the BMI-TIDE® 2× performance assay are provided. The Table providesperformance indices, which where calculated as described above for thevariants, which show improved performance compared to the WT enzyme.Those variants, which have a performance index greater than 1, have animproved performance.

In Table 13.2, data obtained for selected multiple-substitution variantsin the BMI-TIDE® 2× performance assay are provided. The Table providesperformance indices, which where calculated as described above for thevariants, which show improved performance compared to the WT enzyme.Those variants, which have a performance index greater than 1, have animproved performance.

TABLE 13.1 Performance Assay Results for All Variants with PerformanceIndex >1 BMI Tide 2X Variant Liquid Detergent Code [Perf. Index] T004H1.07 T004I 1.25 T004K 1.62 T004L 1.01 T004M 1.05 T004N 1.03 T004P 1.18T004R 1.65 T004V 1.18 T004W 1.21 T004Y 1.32 G012I 1.24 G012K 1.64 G012L1.25 G012M 1.11 G012Q 1.09 G012R 1.54 G012T 1.38 G012V 1.18 G012W 1.46T014F 1.17 T014G 1.17 T014I 1.28 T014K 1.53 T014L 1.19 T014M 1.11 T014P1.04 T014Q 1.24 T014R 1.48 T014S 1.07 T014V 1.14 T014W 1.17 T014Y 1.11E022K 1.79 S023F 1.30 S023I 1.20 S023K 1.67 S023L 1.27 S023M 1.04 S023P1.23 S023Q 1.22 S023R 1.75 S023V 1.09 S023W 1.41 S023Y 1.06 G024F 1.26G024H 1.33 G024I 1.24 G024K 1.70 G024L 1.23 G024M 1.14 G024N 1.28 G024P1.18 G024R 1.67 G024T 1.07 G024V 1.12 G024W 1.42 G024Y 1.12 K033H 1.01Q045F 1.25 Q045H 1.25 Q045I 1.40 Q045K 1.64 Q045L 1.24 Q045N 1.07 Q045R1.68 Q045T 1.09 Q045W 1.60 N046A 1.06 N046F 1.11 N046G 1.07 N046H 1.32N046K 1.61 N046L 1.14 N046M 1.13 N046Q 1.22 N046R 1.19 N046S 1.02 N046T1.20 N046W 1.24 N046Y 1.21 R047K 1.15 Y049F 1.06 Y049I 1.08 Y049K 1.10Y049L 1.06 Y049R 1.54 Y049W 1.34 N050F 1.38 N050H 1.13 N050I 1.36 N050K1.65 N050L 1.35 N050M 1.05 N050P 1.12 N050Q 1.16 N050R 1.81 N050W 1.46N050Y 1.27 T054F 1.16 T054G 1.12 T054H 1.17 T054I 1.34 T054K 1.47 T054L1.26 T054N 1.25 T054Q 1.23 T054R 1.46 T054S 1.03 T054V 1.11 T054W 1.22T054Y 1.08 S058H 1.03 S058N 1.12 S058Q 1.08 T059G 1.11 T059H 1.32 T059I1.43 T059K 1.60 T059L 1.31 T059M 1.10 T059N 1.16 T059P 1.19 T059Q 1.31T059R 1.56 T059V 1.13 T059W 1.32 T060F 1.07 T060I 1.09 T060K 1.49 T060L1.13 T060N 1.07 T060Q 1.10 T060R 1.42 T060V 1.13 T060W 1.23 T060Y 1.07T065F 1.06 T065H 1.07 T065I 1.12 T065K 1.32 T065L 1.10 T065M 1.09 T065P1.11 T065Q 1.01 T065R 1.28 T065V 1.15 T065Y 1.09 S066F 1.05 S066H 1.06S066I 1.24 S066K 1.44 S066L 1.09 S066N 1.00 S066Q 1.12 S066R 1.47 S066V1.19 S066W 1.21 S066Y 1.06 Q087H 1.06 Q087I 1.17 Q087K 1.30 Q087L 1.07Q087M 1.00 Q087N 1.06 Q087R 1.35 Q087T 1.08 Q087V 1.04 Q087W 1.15 N090F1.05 N090H 1.09 N090K 1.37 N090L 1.18 N090R 1.37 N096G 1.00 N096H 1.04N096K 1.54 N096R 1.06 K097H 1.03 K097Q 1.05 K097W 1.02 K100R 1.26 R110K1.05 D119E 1.05 D119H 1.16 D119I 1.09 D119L 1.21 D119Q 1.17 D119R 1.14D119S 1.10 D119T 1.23 D119V 1.24 D119W 1.09 G128F 1.10 G128H 1.27 G128K1.90 G128L 1.20 G128M 1.11 G128N 1.23 G128Q 1.22 G128R 1.94 G128W 1.48G128Y 1.42 S129A 1.12 S129F 1.11 S129G 1.03 S129H 1.17 S129K 1.33 S129L1.01 S129R 1.37 S129T 1.04 S129V 1.01 S129W 1.28 S129Y 1.25 F130I 1.03F130K 1.26 F130R 1.37 F130Y 1.31 S135P 1.03 M138K 1.36 M138Q 1.03 M138V1.10 V140C 1.03 Q151I 1.02 E152A 1.14 E152C 1.15 E152D 1.14 E152F 1.09E152G 1.03 E152H 1.15 E152L 1.15 E152M 1.12 E152N 1.11 E152R 1.19 E152S1.02 E152W 1.06 N155D 1.13 N155K 1.05 N155R 1.14 T179A 1.03 T179F 1.15T179H 1.20 T179I 1.21 T179K 1.62 T179L 1.20 T179M 1.12 T179N 1.04 T179P1.04 T179Q 1.23 T179R 1.49 T179S 1.02 T179V 1.12 T179W 1.05 T179Y 1.07V190H 1.02 V190I 1.16 V190K 1.75 V190Q 1.23 V190R 1.67 S191F 1.18 S191G1.03 S191H 1.29 S191I 1.12 S191K 1.58 S191L 1.07 S191N 1.13 S191Q 1.13S191R 1.74 S191W 1.16 L198M 1.19 L198V 1.05 S199F 1.10 S199I 1.08 S199K1.64 S199L 1.15 S199N 1.14 S199Q 1.14 S199R 1.68 S199V 1.06 Y204H 1.03G205F 1.13 G205H 1.61 G205L 1.14 G205M 1.14 G205N 1.39 G205R 2.07 G205S1.25 G205Y 1.21 K211R 1.23 K214R 1.19 L216F 1.13 L216H 1.05 L216Q 1.05L216R 1.64 L216Y 1.02 N218K 1.57 N218P 1.27 D220E 1.05 D220H 1.00 D220N1.04 D220P 1.05 A221F 1.13 A221I 1.14 A221K 1.49 A221L 1.05 A221M 1.01A221N 1.05 A221V 1.14 A221Y 1.17 G222H 1.01 G222N 1.01 G222R 1.04 Y224F1.05 Y224H 1.29 Y224N 1.23 Y224R 1.12 T243G 1.13 T243H 1.48 T243I 1.06T243K 1.87 T243L 1.11 T243Q 1.36 T243R 1.62 T243W 1.25 T243Y 1.14 V260A1.69 V260D 1.59 V260E 1.17 V260G 2.00 V260H 1.36 V260I 2.09 V260K 1.45V260L 1.18 V260M 1.41 V260P 1.45 V260Q 1.73 V260R 1.47 V260S 1.59 V260T1.66 V260W 1.83 V260Y 1.20 T263H 1.06 S265K 1.30 S265N 1.04 S265R 1.28S265W 1.00 A273I 1.19 A273K 1.47 A273L 1.14 A273N 1.10 A273Q 1.00 A273R1.78 A273Y 1.07 L282F 1.09 L282G 1.14 L282H 1.17 L282I 1.23 L282K 1.67L282M 1.01 L282N 1.08 L282Q 1.17 L282R 1.41 L282V 1.22 S285K 1.20 S285R1.23 Q286K 1.22 Q286R 1.14 A289K 1.23 A289R 1.32 A293R 1.36 N296K 1.28N296R 1.42 A297K 1.56 A297N 1.02 A297Q 1.02 A297R 1.50 G299N 1.02

TABLE 13.2 BMI Performance Assay Results for All Variants withPerformance Index > 1 BMI TIDE ® 2X Liquid Variant Detergent Code [Perf.Index] S023W-G024M 2.36 T004V-S023W-G024W 2.25 S023W-G024Y-A288V 2.14T004L-S023W-G024Y 2.09 N046Q-N050F-T054L 2.03 N050Y-T059R-S129Q 1.97S023W-G024W 1.97 A273H-S285P-E292G 1.94 S023Y-G024Y 1.93 S023Y-G024W1.92 T004S-S023Y-G024W 1.91 N046Q-T054K 1.90 S023W-G024Y 1.90T004V-S023W 1.89 T059K-S066N 1.88 N046Q-N050W-T054H- 1.87 T153AT004V-S023W-G024Y 1.85 L282M-Q286P-A289R 1.83 N046Q-R047K-N050Y- 1.82T054K L044Q-T263W-S285R 1.81 T004L-S023W-G024W 1.79 R047K-N050F-T054K1.78 A273H-S285R 1.78 N050Y-T059K-S066Q 1.78 T054K-Q192K 1.76N046Q-N050W 1.75 L282M-Q286K 1.75 T059K-S066Q 1.74 T004S-S023W 1.74L282M-Q286R-A289R- 1.73 K11N L282M-A289R 1.73 N046Q-N050W-T054H 1.73T059K-S129Q 1.72 T004S-S023N-G024Y- 1.71 F210L T004V-S023W-G024M- 1.70A289V L282M-Q286K-A289R- 1.70 S132T N050W-T054H 1.70 L282M-Q286R 1.69L282F-Q286K-A289R 1.69 T059R-S066Q 1.68 R047K-N050W-T054H 1.68S265P-L282M-Q286K- 1.66 A289R L282M-Q286R-T229S 1.66 L282F-Q286K 1.66T263W-S285R 1.65 S265P-L282M-Q286K 1.65 T263H-A273H-S285R 1.65T059R-S129V 1.64 S032T-T263H-A273H- 1.64 S285R T059R-S066Q-S129Q 1.64T004S-G024W 1.64 T004V-S023W-G024M 1.64 T059K-S066Q-S129Q 1.63L282M-Q286K-A289R- 1.63 I253V T004V-S023Y-G024W 1.63 T059R-S066N-S129Q1.62 N050F-T054L 1.62 T004S-S023N-G024W 1.62 T059R-S066N 1.62T059R-S066N-S129V 1.60 Q286R-A289R 1.60 N046Q-R047K-N050F- 1.60 T054KS265P-L282M-Q286R- 1.57 A289R S265P-L282M-Q286P- 1.68 A289RQ062K-S066Q-S129I 1.59 S023N-G024W 1.59 N046Q-R047K-N050W- 1.58 T054HR047K-T054K 1.58 T004L-G024W 1.58 T014M-T059R-S129V 1.58T059R-S066Q-N092S- 1.58 S129I R047K-N050W-T054K 1.58 T004V-G024W 1.58N047K-N050F-T054K 1.57 S265P-L282F-Q286K- 1.57 N061Y L282F-Q286K-E159V1.57 T004V-S023Y-G024M 1.57 S265P-L282F-A289R- 1.55 T065ST059K-F063L-S066N- 1.55 S129V T004L-S023W 1.55 N050F-T054H 1.55T059R-S066Q-S129V 1.54 V190I-D220E-S265W- 1.54 L282F T004S-S023Y-G024M1.53 T004L-S023N-G024Y 1.53 T059K-S066N-S129I 1.53 T059R-S066N-S129I1.53 L282M-Q286R-A289R- 1.52 P162S N046Q-N050F-T179N 1.52T059K-Y082C-S129V 1.52 T059K-S129I 1.52 N050Y-T054K 1.51T059K-S066Q-V102A- 1.51 S129Q T059R-S066Q-S129I 1.51 T059W-S066N-S129V-1.51 S290R T059R-S129I 1.50 T059K-S066Q-S129I 1.50 T059K-S066Q-S129V1.50 S265P-L282M-Q286R- 1.49 A289R-T202S-K203N T004V-S023N-G024W 1.49S265P-Q286K 1.49 S265P-L282F-A289R 1.49 D220P-S265W 1.49L055F-T059W-S129V 1.49 T059R-S129Q-S191R 1.49 N050W-T054K 1.49T004S-S023W-G024M 1.49 R047K-N050F-T054H 1.48 T059K-S066N-K088E 1.48T059K-S066Q-S129I- 1.48 V291L L282M-Q286R-A289R 1.48 T059R-S066N-F085S-1.47 S129I L282F-Q286P-A289R 1.45 L282F-Q286R-A289R 1.47G099D-S265P-L282F- 1.46 Q286K-A289R N046Q-N050F 1.46 N050Y-T059W-S066N-1.45 S129V T009I-D220P-S265N 1.45 V190F-D220P-S265W 1.45N157Y-T263W-A273H- 1.44 S285R T263W-A273H-S285R 1.44 T263W-S285W 1.44T004V-S023Y 1.43 N046Q-R047K-N050W 1.42 N050W-T054L 1.42N200Y-S265P-L282F- 1.42 Q286P-A289R T059R-S066Q-P264Q 1.42 T004V-G024Y1.40 T004L-G024Y 1.40 N050Y-S191I 1.39 N050Y-T054L 1.39T004L-S023W-G024Y- 1.39 N155K F169I-L282F-Q286R- 1.39 A289RL282M-Q286K-A289R 1.38 F130L-M138L-E152W- 1.38 D183N N046Q-R047K-N050Y-1.38 T054H T004V-G024M 1.38 N050Y-T059W-S066Q- 1.37 S129V S023N-G024Y1.37 T054H-P162Q 1.37 T004S-S023W-G024Y 1.37 N050Y-T054H 1.36L282F-Q286R-A289R- 1.35 F169I R047K-N050W 1.35 V190F-D220P 1.35L282M-F173Y 1.34 T004L-S023Y 1.33 N050W-A288D 1.33 V190I-D220P-S265Q1.33 S265P-L282F-Q286P- 1.24 A289R S265P-L282F-Q286R- 1.39 A289RN046Q-N050Y-T054K 1.33 T059W-S066Q 1.31 T263W-A273H-S285R 1.44T263W-A273H-S285W 1.27 S023Y-G024M 1.30 T004L-S023N-G024W 1.30T004V-S023N-G024Y 1.30 T059W-S066N-S129Q 1.30 T004S-S023Y 1.29T004S-S023N-G024M 1.29 T059W-S066N-A070T 1.29 T059W-S066Q-S129Q 1.29T263W-A273H 1.29 A273H-S285P 1.28 N046Q-R047K-N050Y- 1.28 T054LN046Q-R047K-N050Y 1.28 R047K-N050Y 1.27 T263H-S285W 1.26 R047K-N050F1.25 N046Q-R047K-N050F- 1.25 T054H S023N-G024M 1.25 T004S-G024Y 1.24R047K-N050Y-T054H 1.24 T059W-S066N-S129I 1.22 R047K-T054L 1.21T004S-S023W-G024W 1.21 M138L-E152F-T146S 1.21 D220P-S265N 1.21T004S-G024M 1.20 T004V-S023N 1.20 N046Q-N050F-T054K 1.19N046Q-N050Y-T054H 1.19 Q062H-S066Q-S129Q 1.19 T059W-S129Q 1.19T059W-S129V 1.19 N050F-T054K 1.18 R047K-N050F-T054L 1.18V190I-D220P-S265W 1.18 N112I-T263H-A273H- 1.17 S285R T059W-S066N-S129V1.17 T059W-S066Q-S129I 1.17 T059W-S129I 1.17 T263W-S285P 1.17V190I-D220P 1.16 A289V-T263H-A273H 1.16 T263H-A273H-S285P 1.16N90S-A273H-S285P 1.15 R047K-N050Y-T054L 1.15 T004S-S023N 1.15T059R-S129Q 1.14 N046Q-R047K-T054H 1.14 T059W-S066Q-S129V 1.13E152W-T179P 1.13 N050Y-S066Q-S129V 1.13 T202S-T263W-A273H 1.13T263W-A273H-S285P 1.13 M138L-E152W-T179P 1.11 N046Q-R047K 1.10N046Q-T054H-F176L 1.10 T004L-G024M 1.10 T004S-L282M 1.10 T263H-A273H1.10 T263H-A273H-S285W 1.10 T004L-S023Y-G024M 1.09 L282F-Q286P 1.09T004V-S023Y-G024Y 1.09 V190F-S265W 1.09 M138L-E152F 1.08V190F-D220E-S265W 1.07 N046Q-N050F-T054H 1.06 N157Y-S285W 1.06T004F-S023Y-G024M 1.06 T004V-S023N-G024M 1.06 L198I-D220E-S265Q 1.05N046Q-N050Y-T054K- 1.05 A154T S016L-D220E-S265W 1.05 D220E-S265W 1.04D220E-A237S-S265W 1.04 S066Q-S129Q 1.04 V190F-D220E-S265Q- 1.04 T267IL282M-F173Y-T219S 1.04 E152F-T179P 1.04 V190I-S265W 1.03 M138L-S066Q1.01 M138L-E152W 1.01 T059W-S066Q-A070T- 1.01 S129I V190F-D220E-S265N1.01 V190F-S265N 1.01 N046Q-N050Y 1.01 M138L-E152F-T179P 1.00

Example 14 Stability of nprE Variants

In this Example, experiments conducted to determine the stability ofnprE variants are described. In these experiments, the methods describeprior to Example 1 were used to determine the performance indices (See,“NprE stability assays in the presence of detergent” above). Thefollowing tables provide the results for those variants with PerformanceIndices greater than one (PI>1) tested with and without DTPA.

The stability was measured by determining AGLA activity before and afterincubation at elevated temperature. The table contains the relativestability values compared to WT under these conditions. It is thequotient of (Variant residual activity/WT residual activity). A valuegreater than one indicates higher stability in the presence ofdetergent. In Tables 14.1 and 14.2, data are provided showing therelative stability data of single-substitution variants of NprE relativeto the stability of the WT NprE stability under these conditions, withand without DTPA.

In Tables 14.3 and 14.4, data are provided showing the relativestability data of multiple-substitution variants of NprE relative to thestability of the WT NprE stability under these conditions, with andwithout DTPA.

TABLE 14.1 Stability Results in the Presence of 25% TIDE ® 2X with DTPAStability in the presence of Variant 25% Tide 2x Code with DTPA T004C1.19 T004E 1.05 T004L 1.13 T004S 1.00 G012D 1.06 G012E 1.06 K013A 1.39K013C 1.57 K013D 1.09 K013F 1.30 K013G 1.41 K013H 1.34 K013I 1.33 K013L1.56 K013M 1.28 K013N 1.39 K013Q 1.34 K013S 1.35 K013T 1.22 K013V 1.40K013Y 1.34 S023A 1.01 S023D 1.08 S023F 1.05 S023G 1.11 S023I 1.05 S023K1.07 S023L 1.04 S023M 1.11 S023N 1.09 S023Q 1.03 S023R 1.10 S023S 1.45S023T 1.06 S023V 1.05 S023W 1.08 S023Y 1.15 G024A 1.01 G024D 1.05 G024F1.08 G024G 1.46 G024H 1.05 G024K 1.08 G024L 1.06 G024M 1.10 G024N 1.11G024R 1.07 G024S 1.02 G024S 1.02 G024T 1.04 G024W 1.11 G024Y 1.08 Q045D1.02 Q045E 1.28 N046C 1.29 N046E 1.35 N046Q 1.07 R047K 1.09 R047L 1.13R047M 1.00 R047S 1.21 N050D 1.04 N050F 1.07 N050P 1.03 N050W 1.04 N050Y1.04 T054C 1.04 T054D 1.04 T054E 1.03 T054F 1.03 T054H 1.11 T054I 1.04T054K 1.11 T054L 1.08 T054M 1.06 T054N 1.07 T054Q 1.03 T054R 1.04 T054S1.05 T054V 1.01 T054W 1.07 T054Y 1.07 T059A 1.04 T059C 1.04 T059E 1.02T059G 1.13 T059H 1.07 T059I 1.01 T059K 1.16 T059M 1.10 T059N 1.15 T059P1.12 T059Q 1.04 T059R 1.28 T059S 1.04 T059W 1.26 T060N 1.03 T065E 1.01S066C 1.36 S066D 1.42 S066E 1.58 S066N 1.01 S066Q 1.01 Q087D 1.25 Q087E1.32 N090C 1.10 N090D 1.01 K100H 1.09 K100P 1.01 R110A 1.17 R110C 1.28R110E 1.20 R110H 1.12 R110K 1.04 R110L 1.23 R110M 1.23 R110N 1.11 R110Q1.28 R110S 1.10 R110Y 1.12 D119H 1.15 G128C 1.00 S129A 1.06 S129C 1.38S129D 1.23 S129H 1.30 S129I 1.68 S129K 1.05 S129L 1.35 S129M 1.33 S129Q1.44 S129T 1.36 S129V 1.55 S129Y 1.06 F130I 1.14 F130K 1.04 F130L 1.52F130M 1.66 F130Q 1.10 F130T 1.41 F130V 1.06 S137A 1.46 M138L 1.43 E152F1.15 E152H 1.36 E152W 1.31 T179P 1.50 V190I 1.68 V190L 1.93 S199C 1.27S199E 1.95 Y204T 1.03 K211A 1.96 K211C 1.30 K211D 1.89 K211M 1.20 K211N1.29 K211Q 2.00 K211S 1.43 K211T 1.18 K211V 1.52 K214A 1.74 K214C 1.62K214I 1.17 K214M 1.27 K214N 1.35 K214Q 2.09 K214V 2.00 L216C 1.35 T219D1.05 D220A 1.11 D220E 2.44 D220P 2.66 A221D 1.04 A221E 1.57 G222C 1.72T243C 1.30 T243I 1.17 K244A 1.61 K244C 1.75 K244D 2.00 K244E 1.77 K244F1.27 K244G 1.23 K244L 1.55 K244M 1.79 K244N 1.25 K244Q 1.82 K244S 1.87K244T 1.65 K244V 1.82 K244W 1.01 K244Y 1.45 V260E 1.07 V260K 1.17 V260L1.28 V260M 1.21 V260P 1.22 V260S 1.00 V260T 1.03 V260W 1.02 Y261C 1.28Y261F 1.07 Y261I 1.20 Y261L 1.14 T263E 1.12 T263F 1.19 T263H 1.01 T263L1.02 T263Q 1.12 T263V 1.25 T263W 1.40 T263Y 1.06 S265A 1.04 S265C 1.11S265D 1.11 S265E 1.34 S265P 1.72 S265Q 1.00 S265T 1.15 S265V 1.17 K269E1.61 K269F 1.21 K269G 1.32 K269H 1.63 K269I 1.73 K269L 1.53 K269M 1.52K269N 1.60 K269P 1.47 K269Q 1.55 K269S 1.51 K269T 1.89 K269V 1.43 K269W1.00 K269Y 1.38 A273C 1.19 A273D 1.29 A273H 1.14 R280A 1.33 R280C 1.96R280D 1.82 R280E 1.77 R280F 1.46 R280G 1.21 R280H 1.52 R280K 1.14 R280L1.78 R280M 1.78 R280S 1.46 R280T 1.35 R280W 1.51 R280Y 1.56 L282F 1.06L282M 1.16 L282Y 1.04 S285A 1.16 S285C 1.27 S285D 1.39 S285E 1.59 S285K1.00 S285P 1.30 S285Q 1.10 S285R 1.38 S285W 1.28 Q286A 1.04 Q286D 1.08Q286E 1.31 Q286K 1.09 Q286P 1.15 Q286R 1.18 A289C 1.24 A289D 1.04 A289E1.15 A289L 1.05 A293C 1.11 N296D 1.11 N296E 1.87 N296V 1.37 A297C 1.07

TABLE 14.2 Stability of Variants in Tide ® 2X Without DTPA Stability inthe presence of Variant Tide 2x without Code DTPA T004C 1.16 T004V 1.04K013A 1.52 K013C 1.83 K013D 1.47 K013F 1.02 K013G 1.61 K013H 1.62 K013I1.19 K013L 1.54 K013M 1.48 K013N 1.70 K013Q 1.55 K013S 1.56 K013T 1.39K013V 1.49 K013Y 1.39 S023A 1.03 S023D 1.23 S023G 1.25 S023M 1.05 S023N1.25 S023Q 1.10 S023S 1.50 S023W 1.02 S023Y 1.07 G024D 1.05 G024G 1.41Q045C 1.01 Q045D 1.02 Q045E 1.41 Q045M 1.01 N046C 1.53 N046E 1.41 R047K1.12 R047L 1.20 R047M 1.08 R047Q 1.13 R047S 1.25 Y049D 1.16 Y049H 1.02Y049N 1.07 Y049S 1.01 N050D 1.08 N050F 1.07 N050G 1.02 N050P 1.23 N050W1.01 T054C 1.07 T054D 1.01 T054E 1.08 T054H 1.08 T054I 1.09 T054K 1.03T054L 1.09 T054Q 1.09 T054V 1.14 T054W 1.02 T054Y 1.14 T059A 1.05 T059C1.07 T059E 1.25 T059M 1.04 T059P 1.18 T059Q 1.05 T059S 1.09 T065C 1.04T065E 1.07 S066C 1.61 S066D 1.61 S066E 1.80 S066N 1.08 Q087D 1.27 Q087E1.30 N090C 1.09 N090D 1.00 N090E 1.03 K100A 1.00 K100D 1.07 K100E 1.03K100F 1.07 K100H 1.16 K100N 1.06 K100P 1.06 K100Q 1.06 K100S 1.05 K100Y1.10 R110A 1.11 R110C 1.24 R110E 1.19 R110H 1.09 R110K 1.08 R110L 1.11R110M 1.12 R110N 1.18 R110Q 1.25 R110S 1.09 R110Y 1.16 D119H 1.03 G128C1.15 S129A 1.13 S129C 1.86 S129D 1.52 S129H 1.60 S129I 2.32 S129K 1.18S129L 1.70 S129M 1.64 S129Q 1.86 S129T 1.59 S129V 2.34 S129Y 1.28 F130I1.18 F130L 1.29 F130M 1.44 F130Q 1.17 F130T 1.32 F130V 1.05 S137A 1.37M138L 1.11 E152A 1.01 E152C 1.16 E152F 1.32 E152H 1.53 E152N 1.12 E152W1.32 N155Q 1.07 T179P 1.33 V190I 1.37 V190L 1.40 S199C 1.18 S199D 1.11S199E 1.71 K211A 1.77 K211C 1.18 K211D 1.67 K211G 1.06 K211M 1.17 K211N1.44 K211Q 1.51 K211S 1.44 K211T 1.17 K211V 1.26 K214A 1.47 K214C 1.54K214E 1.42 K214I 1.14 K214M 1.19 K214N 1.15 K214Q 1.84 K214V 1.79 L216C1.31 D220A 1.07 D220E 2.23 D220P 2.24 A221D 1.15 A221E 1.47 G222C 1.89T243C 1.34 T243I 1.13 K244A 1.57 K244C 1.40 K244D 1.58 K244E 1.56 K244F1.05 K244G 1.01 K244L 1.38 K244M 1.37 K244N 1.18 K244Q 1.42 K244S 1.55K244T 1.51 K244V 1.42 K244Y 1.19 V260K 1.09 V260L 1.08 V260P 1.12 V260Y1.02 Y261I 1.19 Y261L 1.11 T263F 1.11 T263H 1.03 T263M 1.08 T263Q 1.04T263V 1.22 T263W 1.37 T263Y 1.05 S265C 1.03 S265D 1.02 S265E 1.22 S265N1.07 S265P 1.43 S265T 1.10 S265V 1.09 K269E 1.33 K269F 1.10 K269G 1.17K269H 1.52 K269I 1.34 K269L 1.34 K269M 1.34 K269N 1.25 K269P 1.26 K269Q1.39 K269S 1.50 K269T 1.32 K269V 1.39 K269Y 1.38 A273C 1.12 A273D 1.16A273H 1.10 R280A 1.32 R280C 1.77 R280D 1.52 R280E 1.67 R280F 1.37 R280G1.16 R280H 1.31 R280K 1.07 R280L 1.64 R280M 1.60 R280S 1.46 R280T 1.28R280V 1.10 R280W 1.42 R280Y 1.49 L282M 1.03 S285A 1.03 S285C 1.10 S285D1.25 S285E 1.36 S285P 1.14 S285Q 1.05 S285R 1.10 S285W 1.12 Q286D 1.05Q286E 1.17 Q286P 1.04 Q286R 1.02 A289C 1.05 A289E 1.13 A289L 1.06 N296C1.01 N296D 1.02 N296E 1.67 N296V 1.32 A297C 1.02

TABLE 14.3 Stability Assay Results in the Presence of 25% TIDE ® 2X WithDTPA Stability in the presence of TIDE ® 2X with DTPA Variant Code[Perf. Index] V190I-D220P 3.08 V190I-D220P-S265Q 2.63 V190L-D220E 2.59V190I-D220E-S265Q 2.57 V190I-D220E- 2.52 S265W-L282F V190L-D220E-S265Q2.43 V190I-D220E-S265W 2.38 V190L-D220E-S265N 2.34 T059R-S066Q-S129I2.33 V190I-D220E-S265N 2.32 V190L-D220E-S265W 2.30 V190I-D220E 2.29T059W-S066N-S129V 2.28 T059K-S066Q-S129V 2.27 T059K-Y082C-S129V 2.27T059R-S066N-S129I 2.27 S066Q-S129V 2.25 T059R-S066Q-S129V 2.25T059R-S129I 2.24 N050Y-T059W- 2.21 S066N-S129V D220P-S265N 2.21S066Q-S129I 2.21 T059W-S066Q-S129V 2.20 T059K-S066Q-S129I 2.20T059R-S129V 2.19 N050Y-S066Q-S129V 2.19 T059W-S066Q-S129I 2.19N050Y-T059W- 2.18 S066Q-S129V T059K-S129I 2.17 D220P-S265W 2.17F130L-M138L-T179P 2.16 S066N-S129I 2.15 T059R-S066N-S129V 2.15F130I-M138L-T179P 2.14 T059R-S066Q-N092S- 2.13 S129I S066N-S129V 2.11D220E-S265Q 2.11 F130L-M138L- 2.10 E152W-T179P T059W-S129V 2.10S265P-L282M- 2.09 Q286R-A289R S265P-L282F-Q286R- 2.09 A289RT059W-S066N-S129I 2.08 V190I-D220P-S265W 2.08 F130L-E152W-T179P 2.06F130L-M138L-E152F- 2.06 T179P Q062K-S066Q-S129I 2.04 T059K-S066N-S129I2.04 E152H-T179P 2.03 S265P-L282M- 2.03 Q286K-A289R F130L-M138L- 2.02E152H-T179P T263W-A273H- 2.00 S285R D220E-S265N 1.99 F130I-M138L-E152H-1.99 T179P F130V-M138L- 1.99 E152W-T179P F130I-M138L- 1.99 E152W-T179PT059W-S129I 1.97 D220E-S265W 1.97 F130V-M138L-T179P 1.96F130L-E152V-T179P 1.96 T059R-S129Q 1.95 T263W-S285P 1.94F130I-M138L-E152F- 1.93 T179P E152W-T179P 1.93 V190L-S265Q 1.93F130L-E152F-T179P 1.92 L282M-Q286R- 1.91 A289R-P162S D220P-S265Q 1.91M138L-E152F-T179P 1.91 F130I-E152H-T179P 1.91 M138L-E152W- 1.91 T179PF130L-T179P 1.90 F130L-M138L- 1.90 E152W-T179P-Q286H F130L-M138L-E152H1.89 T263W-A273H- 1.89 S285W S265P-Q286K 1.88 T059W-S066Q-S129Q 1.87T263W-S285R 1.85 T059W-S066N-S129Q 1.83 T263W-S285W 1.83T059R-S066N-S129Q 1.83 S265P-L282M- 1.81 Q286R-A289R- T202S-K203NT059W-S129Q 1.81 Q062H-S066Q-S129Q 1.81 A282M-Q286R- 1.80 A289RV190L-D220E- 1.80 S265N-V291I V190L-S265N 1.80 F130L-M138L- 1.79 E152WN050Y-T059R-S129Q 1.79 F130I-T179P 1.78 T059K-S066Q-S129Q 1.78T059K-S129Q 1.78 S265P-L282M- 1.77 Q286P-A289R S265P-L282F-Q286P- 1.77A289R T263W-A273H-S285P 1.77 S265P-L282M-Q286K 1.76 S016L-D220E-S265W1.76 S066Q-S129Q 1.76 S265P-L282M-Q286P 1.75 L282F-Q286R-A289R 1.75F130V-E152W-T179P 1.74 L044Q-T263W-S285R 1.74 L055F-T059W-S129V 1.74V190L-S265W 1.74 Q286R-A289R 1.74 G99D-S265P-L282F- 1.73 Q286K-A289RF130L-M138L-E152F 1.73 T059R-S066Q-S129Q 1.72 F130L-E152H 1.71S066N-S129Q 1.71 T004S-S023N- 1.71 G024M-K269N S265P-L282M 1.71E152F-T179P 1.71 T059W-S066N- 1.68 S129V-S290R L282F-Q286K-A289R 1.67F130L-M138L 1.66 F130I-M138L-E152W 1.65 S265P-L282F 1.65F130I-M138L-E152H 1.65 F130V-M138L-E152H 1.64 V190I-S265Q 1.64M138L-E152M 1.61 S265P-L282F-Q286P 1.59 M138L-E152H 1.59T059K-S066N-K088E 1.59 V190I-S265W 1.59 F130L-E152W 1.59 L282M-Q286K-1.58 A289R L282M-Q286K- 1.57 A289R-I253V T263W-A273H 1.56 V190I-S265N1.55 M138L-E152W 1.55 A273H-S285R 1.52 F130I-M138L 1.51 F130L-E152F 1.50F130V-M138L- 1.50 E152W T059K-S066Q- 1.48 V102A-S129Q F130V-E152H-T179P1.47 F130I-M138L-E152F 1.47 F130V-M138L-E152F 1.44 M138L-E152F 1.44L282M-Q286R 1.43 F130I-E152H 1.43 S265P-L282F-A289R- 1.43 T065ST263H-A273H-S285R 1.43 F130V-M138L 1.42 T014M-T059R-S129V 1.42L282M-Q286R- 1.41 A289R-K11N A273H-S285P 1.41 L282M-Q286K- 1.40A289R-S132T T263H-A273H- 1.39 S285W F130V-E152W 1.38 S265P-L282F-Q286K-1.37 N061Y F130I-E152W 1.36 L198I-D220E-S265Q 1.36 V190I-S265L 1.36T263H-S285W 1.35 S265P-L282F-A289R 1.34 M138L-S066Q 1.32 F130I-E152F1.32 N90S-A273H-S285P 1.31 S032T-T263H- 1.31 A273H-S285RL282F-Q286P-A289R 1.28 N157Y-T263W- 1.27 A273H-S285R V105A-S129V 1.26T263H-A273H-S285P 1.25 S129Q-L282H 1.23 T059W-S066Q 1.23 F130V-E152H1.21 S023W-G024Y 1.21 T004V-S023N 1.21 T059R-S066Q 1.21 N050W-T054L 1.20L282M-Q286P-A289R 1.20 A115V-V190L- 1.19 S265W L282M-Q286K 1.19T059R-S066N 1.18 L282F-Q286P 1.15 T004V-S023W- 1.15 G024MS265P-L282F-Q286R- 1.15 L78H L282F-Q286K 1.14 T004V-S023W- 1.14 G024YS023W-G024M 1.13 T059R-R256S 1.13 F130V-E152F 1.12 T004V-G024W 1.12N050W-T054K 1.11 S023Y-G024M 1.11 T004V-S023Y 1.11 T004V-S023Y- 1.11G024M N050Y-T054H 1.10 S023W-G024W 1.10 T004V-S023Y-G024Y 1.10T004V-S023N- 1.09 G024W F130L-M138L-E152F- 1.09 T179P-V291IN050Y-T059K-S066Q 1.09 T004V-S023Y- 1.09 G024W T059K-S066N 1.09T004V-S023N-G024Y 1.09 S023Y-G024W 1.09 N050F-T054L 1.08 R047K-T054K1.08 S023N-G024W 1.07 L282M-A289R 1.07 S023Y-G024Y 1.07 T004V-G024M 1.07L282F 1.06 R047K-N050F-T054K 1.06 N050F-T054K 1.05 T059K-S066Q 1.05S023N-G024M 1.05 S023N-G024Y 1.04 T004L-S023N 1.04 R047K-N050W- 1.04T054H T004L-S023W-G024Y 1.04 T004S-S023W 1.03 N046Q-N050W- 1.03T054H-A142T T004L-S023Y 1.03 T004V-S023W 1.03 N050W-T054H 1.02T004S-S023N 1.02 T004S-L282M 1.02 T004L-S023W 1.02 N050F-T054H 1.01N050Y-T054L 1.00 R047K-N050W- 1.00 T054K

TABLE 14.4 Stability Assay Results in the Presence of 25% TIDE ® 2XWithout DTPA Stability Assay Results in the presence of TIDE ® 2Xwithout DTPA Variant Code [Perf. Index] S066Q-S129V 2.24 S066Q-S129I2.19 N050Y-S066Q- 2.12 S129V S066N-S129I 2.08 T059K-S066Q- 2.06 S129VS066N-S129V 2.05 F130L-E152W- 1.98 T179P S265P-L282M- 1.96 Q286R-A289RF130L-E152V- 1.96 T179P T059K-S066Q- 1.91 S129I T263W-S285P 1.85T059K-S066N- 1.84 S129I T263W-A273H- 1.83 S285P S265P-L282F- 1.83Q286R-A289R F130V-E152W- 1.83 T179P T263W-A273H- 1.82 S285R V190I-D220P-1.79 S265W F130L-E152H 1.78 S066N-S129Q 1.77 S265P-L282M- 1.77Q286K-A289R V190I-D220E 1.76 T059R-S066N- 1.76 S129I V190I-D220E- 1.75S265W T059K-S129I 1.75 T059R-S066Q- 1.75 S129I F130I-M138L- 1.74E152H-T179P F130I-T179P 1.74 T263W-A273H- 1.73 S285W S016L-D220E- 1.72S265W S066Q-S129Q 1.72 V190I-D220E- 1.72 S265Q T059R-S066Q- 1.71 S129VD220E-S265N 1.69 V190L-D220E 1.69 D220E-S265W 1.68 V190I-D220P 1.68V190L-D220E- 1.68 S265N L044Q-T263W- 1.67 S285R S265P-L282M- 1.67Q286P-A289R F130L-M138L- 1.66 E152H-T179P T263W-S285R 1.66 L282M-Q286R-1.65 A289R T263W-S285W 1.65 F130I-E152H- 1.65 T179P V190I-D220E- 1.64S265N V190L-D220E- 1.63 S265W V190I-D220P- 1.63 S265Q T059R-S066N- 1.62S129V V190L-D220E- 1.62 S265Q E152H-T179P 1.62 F130L-M138L- 1.61E152F-T179P Q062H-S066Q- 1.59 S129Q T059R-S129V 1.58 V190I-D220E- 1.58S265W-L282F V190I-S265Q 1.58 F130L-E152F- 1.58 T179P D220E-S265Q 1.57E152W-T179P 1.56 T059K-S066Q- 1.56 S129Q F130L-M138L- 1.55 T179PF130I-M138L- 1.55 E152F-T179P F130L-M138L- 1.54 E152W-T179P N050Y-T059W-1.54 S066Q-S129V S265P-L282M- 1.54 Q286K T059R-S129I 1.53 F130V-E152H-1.53 T179P D220P-S265N 1.52 S265P-L282M- 1.51 Q286P F130I-E152H 1.51T059R-S066Q- 1.51 N092S-S129I F130L-T179P 1.49 G99D-S265P- 1.48L282F-Q286K- A289R T263W-A273H 1.48 V190I-S265N 1.48 D220P-S265W 1.47F130L-E152W 1.47 F130L-M138L- 1.46 E152H S265P-L282M 1.45 V190I-S265Q1.45 F130L-E152F 1.45 T059K-S129Q 1.45 Q286R-A289R 1.45 M138L-E152W-1.44 T179P F130I-M138L- 1.43 E152H D220P-S265Q 1.42 V190L-S265N 1.42F130I-M138L- 1.42 E152W S265P-Q286K 1.41 V190L-S265Q 1.41 V190I-S265W1.40 F130L-M138L- 1.40 E152F F130V-E152H 1.40 E152F-T179P 1.39N050Y-T059W- 1.38 S066N-S129V T059R-S066N- 1.38 S129Q F130I-E152W 1.37F130V-E152W 1.37 T059R-S066Q- 1.37 S129Q T263H-A273H- 1.36 S285PN90S-A273H- 1.36 S285P V190L-D220E- 1.36 S265N-V291I T059R-S129Q 1.35A273H-S285P 1.34 F130I-M138L- 1.34 E152W-T179P F130V-M138L- 1.34 E152FN050Y-T059R- 1.34 S129Q T059W-S066Q- 1.34 S129I F130V-M138L- 1.34 T179PF130V-M138L- 1.33 E152W-T179P V190L-S265W 1.33 F130V-M138L- 1.32 E152WT059W-S066Q- 1.32 S129V V190I-S265Q 1.32 F130V-M138L- 1.32 E152HF130I-E152F 1.31 N157Y-T263W- 1.31 A273H-S285R T263H-S285W 1.30M138L-E152F- 1.30 T179P A115V-V190L- 1.29 S265W M138L-E152M 1.29T263H-A273H- 1.29 S285W F130L-M138L- 1.28 E152W T059K-S066N- 1.28 K088EF130I-M138L- 1.27 E152F F130I-M138L- 1.27 T179P T004V-S023N 1.26T059K-S066Q- 1.26 V102A-S129Q F130L-M138L 1.26 N047K-N050F- 1.24 T054KT263H-A273H- 1.24 S285R F130L-M138L- 1.23 E152W-T179P- Q286H M138L-E152H1.22 M138L-S066Q 1.22 L282M-Q286R- 1.21 A289R-P162S L282F-Q286R- 1.21A289R Q062K-S066Q- 1.21 S129I A273H-S285R 1.20 S265P-L282F- 1.20 Q286PS265P-L282F- 1.20 Q286P-A289R S265P-L282M- 1.19 Q286R-A289R- T202S-K203NT059W-S066N- 1.19 S129I V190I-S265L 1.18 T059W-S066N- 1.18 S129VF130I-M138L 1.16 L282M-Q286K- 1.16 A289R-I253V R047K-N050F- 1.15 T054KM138L-E152F 1.15 N050W-T054K 1.15 L198I-D220E- 1.13 S265Q L282F-Q286K-1.13 A289R N050F-T054K 1.13 L282M-Q286R 1.13 M138L-E152W 1.13S265P-L282F 1.12 F130V-E152F 1.12 T059W-S066N- 1.10 S129Q F130V-M138L1.09 T263H-A273H 1.09 L282M-Q286K- 1.07 A289R N046Q-N050W- 1.07T054H-A142T T059W-S066Q- 1.07 S129Q S265P-L282F- 1.07 A289R-T065SN050F-T054H 1.07 S129Q-L282H 1.06 L282M-Q286K- 1.03 A289R-S132TL282M-Q286R- 1.03 A289R-K11N T059K-S066N 1.02 R047K-N050W- 1.01 T054KT059K-S066Q 1.01 T004V-S023Y 1.01 T059W-S066N- 1.00 S129V-S290RN050Y-T059K- 1.00 S066Q R047K-N050Y 1.00

The data in the following table (Table 14.5) represent the relativestability data of variants of NprE relative to the stability of the WTNprE stability in the citrate stability assay. The stability wasmeasured by determining casein activity by determining AGLA activitybefore and after incubation at elevated temperature (See, “CitrateStability Assay” above). The table contains the relative stabilityvalues compared to WT under these conditions. It is presented as thequotient of (Variant residual activity/WT residual activity). A valuegreater than one indicates higher stability in the presence ofdetergent.

TABLE 14.5 Citrate Stability Assay Results Citrate Variant StabilityCode Relative K013C 1.22 K013D 1.32 K013E 1.07 K013H 1.50 K013Q 1.38K013S 1.11 T014G 1.31 T014H 1.75 T014K 1.62 T014M 1.09 T014P 1.07 T014Q2.01 T014R 1.32 T014V 1.03 S023A 1.12 S023G 1.13 S023I 1.13 S023K 1.39S023M 1.00 S023N 1.42 S023T 1.15 S023V 1.20 S023W 1.22 G024D 1.38 G024F1.90 G024H 1.09 G024M 1.23 G024R 1.03 G024S 1.11 G024T 1.03 G024W 1.03Q045D 1.07 Q045E 1.12 Q045M 1.02 Q045N 1.16 Q045P 1.44 N046G 1.10 N046H1.05 N046I 1.46 N046P 1.47 N046V 1.11 N046Y 1.01 R047E 1.09 R047T 1.07Y049A 1.02 Y049C 1.03 Y049D 1.01 Y049E 1.04 Y049I 1.08 Y049K 1.04 Y049T1.16 Y049V 1.19 Y049W 1.00 T054D 1.01 T054H 1.09 T054K 1.02 T054L 1.06T054P 1.63 T054Q 1.17 T054R 1.11 T054S 1.09 T054W 1.02 S058I 1.23 S058L1.71 S058N 1.08 S058P 2.53 T059E 1.08 T059H 1.19 T059I 1.02 T059K 1.21T059L 1.16 T059M 1.04 T059S 1.07 S066D 1.03 S066E 1.03 S066P 1.13 S066Q1.05 S066T 1.17 S066V 1.00 Q087A 1.05 Q087L 1.08 Q087S 1.15 Q087T 1.19N090D 1.17 N090F 1.02 N090G 1.04 N090L 1.25 N090T 1.02 N096G 1.02 K100D1.30 K100N 1.28 K100P 1.04 K100V 1.01 D119H 1.05 D119T 1.03 D119W 1.00G136I 1.10 G136L 1.20 G136P 2.19 G136V 2.03 G136W 2.23 G136Y 1.56 M138L1.48 D139A 2.52 D139C 2.22 D139E 1.51 D139G 2.54 D139H 1.88 D139I 2.40D139K 2.27 D139L 1.53 D139M 2.49 D139P 2.21 D139R 2.54 D139S 2.22 D139V1.51 D139W 1.94 D139Y 2.54 E152C 1.17 E152F 1.21 E152G 1.09 E152H 1.29E152R 1.12 E152S 1.17 E152W 1.21 D178A 2.07 D178C 1.79 D178G 2.35 D178H2.07 D178K 1.73 D178L 1.74 D178M 2.40 D178N 2.34 D178P 1.83 D178Q 1.22D178R 2.00 D178S 2.58 D178T 1.75 D178V 1.73 D178W 1.02 D178Y 1.78 E186A2.31 E186C 2.42 E186D 2.03 E186G 2.09 E186H 1.87 E186K 2.69 E186L 1.75E186M 2.62 E186N 1.72 E186P 2.60 E186Q 1.92 E186R 2.69 E186S 2.57 E186T2.69 E186V 2.10 E186W 2.47 E186Y 2.48 V190I 1.38 V190L 1.41 K211A 1.33K211M 1.26 K211Q 1.16 K211S 1.28 K214A 1.38 K214C 1.12 K214E 1.08 K214I1.30 K214L 1.14 K214M 1.03 K214Q 1.47 K214R 1.12 K214S 1.05 K214V 1.49L216A 1.05 L216C 1.04 L216S 1.19 D220E 1.69 D220H 1.17 D220K 1.17 D220N1.01 D220P 1.20 A221D 1.11 A221S 1.05 G222C 1.01

Example 15 pH Performance Profile of nprE Compared to BPN′ 217L

In this Example, experiments conducted to evaluate the comparativeperformance of nprE and BPN′ Y217L are described. In these experiments,EMPA 116 (BMI) and Equest grass stains were used.

Materials:

NprE, 8 mg/mL

BPN′ Y217L, 25.6 mg/mL

EMPA 116 soil cloth, 3″×4.5″ (Testfabrics)

Equest grass (med.), 3″×4″ (Warwick Equest)

EMPA 221 white cotton swatches, 3″×4.5″

Minolta Chromameter CR200

TIDE® 2005 (provided by Procter & Gamble)

Water hardness concentrate: 15,000 grains per gallon (gpg), 3:1 Ca:Mg

1 M Bis-TRIS-propane buffer

Conc. sulfuric acid

50 L mix tank with spigot and agitator

Terg-O-Tometer

DI Water

The swatches were prepared for treatment. Three replicates per treatmentwere conducted, with 18 swatches used per treatment. The grass swatcheswere prepared in a dark room to prevent fading. The reflectance valuesof about 18 soiled swatches were obtained using a Minolta Chromameter.Three readings were obtained per swatch. The L values, average L valueand standard deviation were recorded. This is the L_(initial) value.

The detergent solution was prepared as follows (for 40 L total). It waspreferred to prepare this solution the night before testing. Thesolution was stored in the cold over night. The solution was prepared byadding 39.724 Kg of DI water to 50 L mix tank, starting the agitator,mixing in 60 grams of TIDE® liquid detergent, mixing in 16 mL of waterhardness solution, and 200 mL of 1 M Bis-TRIS-propane. The pH wasadjusted using concentrated sulfuric acid (adjusted to 0.2 pH unitsbelow desired pH, if solution was stored overnight). as pH creeps upovernight). The final concentrations were: TIDE®=1.5 g/L; waterhardness=6 gpg; and Bis-TRIS-propane=5 mM.

For testing in the Terg-O-Tometer, 1 L of detergent solution was addedto each Terg pot and allowed to come to temperature. Enzyme was added tothe pots at varying concentrations. For BMI tests, the enzymeconcentrations used were 0 mg/L, 0.275 mg/L, 0.55 mg/ml, 1.65 mg/L, 2.65mg/L, and 5.50 mg/L. For grass stains, the nprE concentrations used were0 mg/L, 0.1925 mg/L, 0.385 mg/L, 1.155 mg/L, 1.925 mg/L, and 3.85 mg/L(the concentrations of BPN′ Y217L were the same as those used in the BMItests). Agitation was started and the swatches were added. Allreplicates were run side-by-side in the same Terg-O-Tometer (e.g., 0× &½× n the 1^(st) run, 1× & 3× in the 2^(nd) run, and 5× & 10× in the3^(rd) run). The temperature was 15° C., the agitation speed was 100cpm, and the wash time was 12 minutes. The treated swatches were rinsedthree times in 4 L tap water (˜6 gpg). The swatches were air-driedovernight on paper towels. The grass swatches were covered with papertowels and allowed to dry in a darkened room. The reflectance values ofthe dried swatches were determined as described above. Three readingswere obtained per swatch. The L values. average L value and standarddeviation were recorded. This is the L_(final) value.

The percentage of soil removal (% SR) was determined for each testingcondition and both enzymes using the equation below:

${\%\mspace{14mu}{SR}} = {\frac{\left( {L_{final} - L_{initial}} \right)}{\left( {L_{0} - L_{initial}} \right)} \times 100\%}$Where:

-   -   L₀=reflectance of unsoiled swatches    -   L_(initial)=reflectance of soiled swatches    -   L_(final)=reflectance of washed swatches

The delta % SR over no enzyme control was determined using the followingformula:Δ% SR=% SR_(treatment)−% SR_(no enzyme control)

BPN′ Y217L was compared to nprE on EMPA 116 (BMI), at pH values of 6.7,7.5, 8.5, and 9.5. The performance of nprE on EMPA 116 appeared to peakat about pH 8, while the performance of BPN′ Y217L peaked at about pH8.8. The results showed that nprE performed better than BPN′ Y217L at pH7.5 and 8.5, although it does not perform as well as BPN′ Y217L at pH6.7. The performance of these enzymes was equal at pH 9.5. In addition,there was no difference in the performance of these enzymes on Equestgrass (med) at pH 7.8-8.4.

Example 16 Comparison of PMN and nprE Enzymes in Liquid Detergent

This Example describes cleaning experiments to determine the cleaningperformance of PMN and nprE. The cleaning performance of PMN and nprEenzymes were tested in Liquid TIDE® detergent in comparison with abenchmark serine protease (Protease A) on protease sensitive stains. Asshown in the table below, PMN and nprE remove stains much better thanprotease A, even at low enzyme levels. In the following Tables, thehigher SRI values indicate a better cleaning performance.

TABLE 16.1 Comparison of Cleaning Performance of PMN vs. Protease A inLiquid TIDE ® (in full size washing machine) Active Enzyme Protein inthe wash solution 0.55 ppm 0.55 ppm 5.50 ppm 5.50 ppm Protease A PMNProtease A PMN SRI on Lightly Soiled 53.1 60.8 60.7 67.2 Grass StainsSRI on Medium Soiled 46.5 55.0 54.2 59.8 Grass Stains SRI on HeavilySoiled 39.1 45.8 44.5 51.6 Grass Stains

TABLE 16.2 Comparison of Cleaning Performance of PMP vs. Protease A inLiquid TIDE ® (in mini size washing machine) Active Enzyme Protein inthe wash solution 0.55 ppm 0.55 ppm Protease A PMN SRI on Lightly SoiledGrass Stains 28.1 52.8 SRI on Medium Soiled Grass 22.8 33.1 Stains SRIon Heavily Soiled Grass Stains 19.9 24.2Table 16.2. Comparison of Cleaning Performance of PMP vs. Protease A inLiquid TIDE® (in Mini Size Washing Machine)

TABLE 16.3 Comparison of Cleaning Performance of nprE vs. Protease A inLiquid TIDE ® (in mini-size washing machine) Active Enzyme Protein inthe wash solution 0.55 ppm 2.75 ppm 5.50 ppm 0.28 ppm Protease AProtease A Protease A nprE SRI on Lightly Soiled 26.3 30.8 30.7 31.5Grass Stains SRI on BMI Stains 19.4 24.9 21.4 25.0 Baby Food Beef Stains63.2 68.8 69.4 71.1

Example 17 Thermostability of NprE and NprE Variants

In this Example, experiments conducted to determine the thermostabilityof NprE and NprE variants are described. The enzymes were produced andpurified as described above. The purified proteins were judged to besufficiently homogenous, with greater than 95% purity as determinedusing 10% SDS-PAGE, as only one major protein was observed in the gel.This protein was approximately 32 kDa, which is the molecular weight ofthe mature nprE sequence. The protein was formulated for storage usingthe 25 mM MES buffer, pH 5.8, containing 1 mM zinc chloride, 4 mMcalcium chloride, and 40% propylene glycol. The assays used in theseexperiments were the protease assay using fluorescence AGLA activitydescribed above and differential scanning calorimetry (DSC), describedbelow.

Differential Scanning Calorimetry (DSC)

Excessive heat capacity curves were measured using an ultrasensitivescanning high throughput microcalorimeter VP-Cap DSC (Microcal). Thestandard procedure for DSC measurements and the theory of the techniqueis well known to those of skill in the art (See e.g., Freire, 1995)Meth. Mol. Biol., 41, 191-218 [1995]). Briefly, approx. 500 uL of200-400 ppm pure or ultrafiltrate concentrate (UFC) protein samples wereneeded. Typically, 400 ppm of NprE and the variant proteins (in theabsence and presence 130 mM citrate) were scanned over 20-100° C.temperature range using a scan rate of 200° C./hr in 5 mM HEPES, pH 8.0buffer. The same sample was then rescanned to check the reversibility ofthe process. For NprE, the thermal unfolding process was irreversible.Scan rate dependence data of the thermal melting for NprE was assessedover a scan rate of 25 to 200° C./hr. The effect of various additives(e.g., primary and secondary alcohols, salts, cyclodextrin, PEG,sorbitol, glycerol) on the thermal melting point of NprE was alsoassessed.

Results

The thermal stability of wild-type NprE was determined at two differentconcentrations, in order to show the effect of protein concentration onthe thermal melting point. The Tm values for 220 ppm and 440 ppm weredetermined to be 67.6±0.5 and 69.2±0.5° C., respectively. The proteinconcentration effect highlights a second-order event. It is contemplatedthat this is either aggregation or autolysis. However, it is notintended that the present invention be limited to any particularmechanism. Nonetheless, these results indicate that for an accuratedetermination and any comparison of thermal melting points for NprErequire that the protein concentrations be well matched. The effect ofthe scan rate on the thermal melting point also showed a dependencewhere the Tm was dependent on the scan rate up to 150° C./hr, and thenleveled off between 150-200° C./hr. Based on these results, 200° C./hrwas selected as the upper scan rate for all studies to minimize thedependence of the Tm on scan rate.

All data collected for NprE and variants are shown in Table 4. Table 4also includes the DSC thermal melting points obtained for NprE andvariants in the presence of 130 mM citrate. In most cases, two proteinconcentrations were scanned. As indicated in this Table, in the case ofthe scans in the presence of 130 mM citrate not all proteins showed athermal unfolding profile.

TABLE 17 DSC Results DSC Thermal Concentration Concentration 440 ppm 220ppm 440 ppm Protein with Enzyme Tested Protein Protein 130 mM citrateWild-type NprE 67.6 +/− 0.5 69.2 +/− 0.5 No transition Thermolysin87.0000 52.1000 B. subtilis NprE 68.0000 55.0000 FNA 64.9000 51.7000T14R 57.0000 51.7000 S23K 67.8000 None S23R 67.8000 53.5000 G24R 63.700050.7 Q45E 70.6000 70.7000 53.0000 N46K 63.8000 50.7 S58D 63.3000 50.5000T59P 68.8000 49.1000 S58D, T60D 59.0000 No transition T60D 66.2000 Notransition S66E 70.3000 71.6000 S129I 70.2000 70.7000 50.3000 S129V69.9000 70.3000 No transition F130L 69.8000 48.5000 M138I 69.200052.5000 M138L 67.8000 V190I 69.0000 69.4000 51.5 L198M 68.2000 68.500053.3000 S199E 70.3000 70.3000 49.1000 D220P 69.3000 69.9000 49.4000D220E 69.4000 69.8000 50.5000 K211V 69.8000 K214Q 68.9000 A221S 59.100052.5000 G222C 69.5000 No transition K244S 67.6000 K269T 69.5000 51.5R280D 67.4000 67.9000 49.2000 N296E 60.5000 69.8000 49.5000 N50W, N296E62.4000 47.2000 G5C, N61C 67.8000 48.4 Q45K, S199E 67.7000 51.3000F130L, D220P 62.7000 70.3000 50.8000 M138L, D220P 63.2000 68.200050.8000 S129I, V190I 70.3000 55.8000 S129V, V190I 69.9000 55.3000 S129V,D220P 70.6000 55.7000 S129I, D220P 70.7000 53.5000 S129V, R280L 69.500054.9000 V190I, D220P 69.8000 52.8000 Q45K, S199E 67.7000 51.3000 N50W,N296E 62.4000 47.2000 G24K K269T D220E 65.0000 51.5000 S129I, F130L,D220P 68.9000 56.6000 nprE-T004S-S023N- 64.6000 None G024M(+K269N)nprE-T004V-S023N 71.2000 49.0000 nprE-S023W-G024Y 64.0000 NonenprE-T004V-S023W- 65.5000 49.3000 G024M nprE-T059K-S66Q-S129I 70.500049.3000 nprE-T059R-S66N-S129I 70.2000 54.0000 nprE-T059R-S129I 69.400054.0000 nprE-T059K-S66Q- 70.3000 56.0000 S129V

A representative Figure of the thermal unfolding profiles (DSC scans)for wild type and various mutants of NprE are shown in FIG. 27. Theunfolding profiles indicate the wild-type midpoint and show selectivemutants that display increased thermal melting points relative towild-type and those that display decreased melting points relative towild-type. This Figure clearly highlights that the DSC distinguishedbetween stable and less stable NprE variants, and is useful as asecondary screen. A general trend is observed between the thermalmelting points of the variants and their stability in detergent. Forexample, the variants S66E, S199E, D220P/E, S129I/V are all winners inTIDE® and show an approximate 1° C. increase in thermal melting pointrelative to wild type NprE. This 1° C. increase in thermal melting pointis small yet significant, as thermal stability typically requiresmultiple amino acid substitutions.

FIG. 28 shows the thermal unfolding of NprE variants that display athermal unfolding profile in the presence of 130 mM citrate. Citrate isa detergent component that rapidly causes the autolysis of NprE, in theabsence of calcium. For wild-type NprE, there is no thermal unfoldingprofile in the presence of citrate, which is consistent with a proteinthat is already unfolded or lacks a well-formed hydrophobic core.Mutants that display a thermal unfolding profile in the presence ofcitrate are included in Table 17. These variants have thermal meltingpoints in the range of 47-56° C. The DSC scans in the presence of 130 mMcitrate indicated variants that are more stable than wild-type NprE tocitrate. For example, citrate-stable variants are show to contain eitherS129I or S129V and combinatorials containing either of thesesubstitutions show a+5° C. increase in thermal melting point.

Effect of Additives on the Thermal Melting Points of NprE:

FIG. 29 shows the results of experiments including various additives.The buffer was 5 mM HEPES, pH 8.0. The samples were scanned from 20-100°C. using a scan rate of 200° C./hr. In this Figure, the horizontal linerepresents the Tm for wild-type NprE with no additive. In theseexperiments, the data showed little or no effect on the thermal meltingpoint (Tm) of NprE in the presence of these reagents. The inclusion ofan inhibitor of NprE activity, namely phosphoramidon, was shown toincrease the Tm by approx. 1° C., suggesting that the inhibitor mayimpart some stabilization to NprE against the thermal unfolding process.None of the conditions above assisted in making the thermal unfoldingprocess reversible. However, it is not intended that the presentinvention be limited to any particular mechanism.

Example 18 NprE Homologue Stability in TIDE® and Homolog BMI WashPerformance

In this Example, experiments conducted to assess the stability of nprEhomologs in TIDE®, as well as the wash performance of these homologs aredescribed. Purified NprE (“NprE”), Bacillus subtilis NprE (B.S. NprE),Bacillus thuringiensis NprB (B.T. NprB) and Bacillus thermoproteolyticusthermolysin (TLN) were incubated in 200 ul 25% tide in 10 mM HEPES, pH8at 10 ug/ml at 25° C. for 90 mins. The initial activities and remainingactivities were measured using the AGLA assay, as described above.Briefly, 10 ul of sample were added into 200 ul of AGLA buffer (50 mMMES, pH6.5, 0.005% TWEEN®-80, 2.5 mM CaCl₂), then 10 ul of dilutedsample was added into 200 ul of AGLA substrate (2.4 mM Abz-AGLA-Nba inAGLA buffer). The excitation at 350 nm and emission at 415 nm weremonitored for first 100 seconds, the initial slope was recorded asenzyme activity. The percent of remaining activity was calculated bydividing the remaining activity over initial activity. FIG. 30 providesa graph showing the remaining activity after 90 minutes.

To determine the wash performance of these homologues in TIDE®, onepre-washed BMI microswatch was first added into each well of a 96-wellplate. Then, 190 ul of 1× compact TIDE® (780 ug/ml compact TIDE®, 5 mMHEPES, pH8, 6gpg water hardness) were added. Then, 10 ul of purifiedNprE, Bacillus subtilis NprE, Bacillus thuringiensis NprB and Bacillusthermoproteolyticus thermolysin were added to the wells to produce afinal enzyme concentration is 0.25 ug/ml. The plate was incubated at 25°C. for 30 mins with shaking at 1400 rpm on Thermomixer. At the end ofincubation, 100 ul of supernatant were transferred into a new 96-wellplate. The OD at 405 nm of supernatant was then measured. Thesupernatant OD was subtracted with the OD of a blank control withoutenzyme addition. The performance index was calculated by dividing the ODof each homologue to the OD of NprE. FIG. 31 provides a graph showingthe BMI was performance of NprE, as well as the nprE homologs describedherein.

Example 19 Metal Analysis of Wild-Type nprE and Variants

In this Example, experiments conducted to determine the zinc and calciumcontent of nprE and nprE variants are described. In these experiments,total trace metal analysis by inductively coupled plasma—massspectrometry (ICPMS) and particle induced X-ray emission with amicrofocused beam (micro-PIXE) were performed to confirm the zinc andcalcium content of NprE. Overall, one zinc and two calcium ions aretightly bound.

All ICPMS and micro-PIXE samples were prepared in metal free buffer toremove any exogenous metal contaminants. Typically, 250 uL of 40 mg/mLNprE samples were buffer exchanged three times with 20 mM HEPES, pH 8.2using YM-10 microdialysis apparatus. Metal free buffer was generated bypassing the buffer through a column packed with Chelax 100 resin. Thefinal protein concentration was determined using Bicinchoninic acidprotein determination assay kit (BCA assay) from Sigma. ICPMS sampleswere analyzed at the West Coast Analytical Services, Inc. Micro-PIXEsamples were analyzed at the Ion Beam Analysis Laboratory.

Table 19-1 shows ICPMS metal analysis results for calcium and zinc ionsfrom NprE wild type. Relative to protein concentration, two calcium ionsand two zinc ions were found to be present in the sample.

TABLE 19-1 ICPMS Metal Analysis of Wild-Type NprE Ca (ppm) Zn (ppm)ICPMS 73.8 156 Mol w (g/mol) 40.08 65.37 Protein (ppm) 833 833Ratio/protein 1.4 1.9

The MicroPIXE elemental composition analysis plot measured the metalcontents relative to a protein internal standard. All peaks detectedusing Micro-PIXE were calculated relative to the sulfur peak arisingfrom three methionines in the case of NprE. An observed large chlorideion peak was due to the presence of salt in buffer.

Table 18-2 shows the metal content determined by Micro-PIXE, whichindicates that in general, NprE contains two tightly bound calcium andone zinc ion per protein molecule. Wild type NprE showed 1 zinc ion with2 calcium ions. It is contemplated that calcium ions may have shown alow occupancy rate due to preparation of the sample. S58D and T60Dshowed close to two zinc ions per protein indicating a possible extrazinc ion binding to the site. The double variant has two added cysteinesadding the accuracy of the technique. However, it is not intended thatthe present invention be limited to any particular embodiment with aspecific number of ions.

TABLE 19-2 Micro-PIXE Metal Determination Showing Ca and Zn Contents forNprE Native and Variants #S Ca/S Ca/prot Zn/S Zn/prot Ca/Zn S58D 3 0.722.2 0.52 1.6 1.4 T60D 3 0.68 2.0 0.57 1.7 1.2 S58D.T60D 5 0.41 2.1 0.221.1 1.9 N46K 3 0.59 1.8 0.42 1.3 1.4 S23K 3 0.62 1.9 0.33 1.0 1.9 A221S3 0.76 2.3 0.5 1.5 1.5 WT 3 0.54 1.6 0.34 1.0 1.6

Consistent with other well-characterized calcium and zinc dependentneutral proteases such as thermolysin or thermolysin-like proteases(TLPs) (See e.g., Dahlquist et al., Biochem., 15:1103-1111 [1976];Srpingman et al., (1995) Biochemistry 34, 15713-15720 [1995]; andWillenbrock et al., (1995) FEBS Lett. 358:189-192 [1995]), NprE wasfound to contain at least two tightly bound calcium ions and one zincion per molecule. A potential third calcium binding site is proposed butexpected to be very weak. Since all samples were desalted to remove anyexogenous metals, these weakly bounding calcium sites are expected to beunoccupied.

Example 20 Stabilizing NprE with Calcium Formate in TIDE® Compact HDLDetergent

In this Example, experiments conducted to develop means to stabilizeNprE in TIDE® compact HDL detergent are described. In these experiments,means to stabilize NprE by increasing the calcium formate level at afixed citrate concentration while lowering DTPA content in experimentalTIDE® compact formulation (“TIDE® 2×”) were investigated. A statisticaldesign of experiments (DOE) methodology was used in order to simplifythe experiments as well as data analyses. It was shown that DTPA presentin TIDE® adversely affects NprE stability, while addition of calciumformate helps overcome the detrimental effect in the full strength TIDE®compact formulation.

A full central composite response surface model with duplicate centerpoints was used as a DOE method. A total of 16 unique formulationsvarying four components were pre-made according to the compositionvariations listed in Table 19.1. LAS was varied from 0-6% (w/w) withDTPA (0-0.25%) and calcium-formate (0-0.2%) at a fixed concentration ofcitric acid (1.9%). All other components of the TIDE® detergent wereheld constant. The component concentration boundary conditions weredetermined based on phase stability of the various mixes. The proteinstability tests were conducted with 780 ppm nprE in the full strength(˜100%) formulation mixes and incubated at 32° C. Inactivation wasmeasured up to 24 hours. All assays were done using red fluorescentlabeled casein assay kit (Invitrogen) with 0.5 ppm proteinconcentration. Rates of NprE inactivation were measured in threeindependent experiments. DOE data were analyzed using DOE Fusion Pro(S-Matrix).

TABLE 20.1 Composition of the 16 TIDE ® Formulations Used for DOEStudies HLAS Citric acid DTPA Ca formate Form 1 3 1.9 0 0.1 Form 2 3 1.90.125 0.1 Form 3 3 1.9 0.25 0.1 Form 4 6 1.9 0.25 0.2

Table 20.2 and FIG. 31 show the results of NprE stability measurementsin various formulation mixes. Average rates and the standard deviationwere the averaged NprE inactivation rate (hour⁻¹) from three independentmeasurements. Qualitatively, formulations with low DTPA content withhigh calcium load tend to be more stable in the full strength compactTIDE®. As an example, Formulation #5, with no addition of DTPA and highcalcium formate level showed the lowest inactivation rate, indicatinghigh NprE stability. In contrast, Formulation #9, with high DTPAconcentration with no added calcium formate showed lowest stability. InTable 20.2, the ranking is based on measured stability (i.e., averagedrates). Runs are from three independent stability experiments.

TABLE 20.2 NprE Inactivation Rates in 16 Formulation Mixes Average RateStandard Ranking Run 1 Run 2 Run 3 (hour⁻¹) Deviation Form 5 1 0.0310.053 0.067 0.050 0.019 Form 1 2 0.060 0.044 0.081 0.062 0.019 Form 15 30.050 0.079 0.060 0.063 0.015 Form 6 4 0.312 0.057 0.059 0.143 0.147Form 7 5 0.364 0.254 0.128 0.249 0.118 Form 11 6 0.099 0.288 0.395 0.2610.150 Form 10 7 0.337 0.238 0.226 0.267 0.061 Form 16 8 0.063 0.5930.188 0.281 0.277 Form 2 9 0.392 0.372 0.296 0.354 0.051 Form 14 100.387 0.451 0.269 0.369 0.093 Form 4 11 0.665 0.333 0.336 0.445 0.191Form 8 12 0.682 0.554 0.378 0.538 0.153 Form 3 13 0.864 0.440 0.3890.566 0.261 Form 13 14 1.417 0.931 0.964 1.104 0.272 Form 12 15 1.0051.620 1.029 1.218 0.349 Form 9 16 0.875 2.099 0.694 1.223 0.764

FIG. 33 shows NprE inactivation effects by DTPA at varying levels offixed calcium formate concentration. Panel A shows rate of NprEinactivation by DTPA without any added calcium formate. The correlationshows that DTPA has significant detrimental effect. Panel B shows somedecreased effect of DTPA with 0.1% calcium formate. Panel C showssignificantly decreased effect of DTPA with 0.2% calcium formate.

FIG. 34 shows DOE analysis software (Fusion Pro) generated predictionprofile of DTPA and calcium formate composition based on response goal(decay rate) of less than 0.5 hr⁻¹ (Panel A), 0.25 hr⁻¹ (Panel B) and0.05 hr⁻¹ (Panel C). The shaded areas indicate DTPA and calcium formatecomposition ratios that are predicted to show stability with decay ratebelow the set goal. For example, 0.16% calcium formate in the presenceof 0.04% DTPA would provide NprE stability with decay rate of less than0.25 hour⁻¹ as shown in Panel B of FIG. 34. On the other hand, 0.08%calcium formate cannot sustain NprE stability with decay rate of atleast 0.25 hour⁻¹ in the presence of 0.16% DTPA.

Example 21 Identification of the Citrate-Induced Autolytic Sites for B.amyloliquefaciens Neutral Metalloprotease NprE

In this Example, methods used to assess the citrate-induced autolysis ofwild-type and recombinant variant nprE (e.g., B. subtilis variant) aredescribed. In these experiments, autolysis of the neutralmetalloprotease from B. amyloliquefaciens (natural and the recombinantvariant expressed in B. subtilis) was induced using sodium citrate(Sigma). The autolysis process was controlled by performing the reactionat 4° C. in 25 mM MES, pH 6.5, buffer. In these experiments, theautolysis of 0.4 mg/ml NprE was optimized by varying either: (a) thetime of incubation (10-100 minutes) in 10 mM citrate; or (b) the citrateconcentration (10-100 mM) over 100 minutes. A control of neutralmetalloprotease diluted in buffer alone (i.e., no citrate) was incubatedunder similar conditions. The autolytic reactions were terminated byaddition of an equal volume of 1N HCl, the samples were precipitatedusing TCA and the pellet was washed and dried using acetone. Theresultant pellet was resuspended in 20 uL buffer, pH 6.5, and 4×LDSsample buffer (NuPage, Invitrogen)

The autolytic fragments were resolved by 10% (w/v) SDS-PAGE andelectroblotted onto a PVDF membrane. The first 10 amino acid residueswere sequenced by Edman degradation (Argo Bioanalytica). The partialamino acid sequences of the autolytic fragments were determined usingtrypsin in-gel digestion and analyzed using LCQ-MS (Agilent). The in-geldigestion process involved macerating the gel piece that contained theprotein, removal of the Coomassie blue stain followed by re-hydration ofthe gel pieces in 25 mM NH₄CO₃ containing 2 M urea. Trypsin was added tothe re-hydrated gel pieces for approx. 6 hours at 37° C. Following thedigestion, the peptides were extracted using acetonitrile and TCA. Thepeptides were separated on a C4-hydrophobic column (Vydac) using anacetonitrile-water gradient. The resultant peptide maps were searchedwith the SEQUEST® database search program against a database containingGenencor enzymes.

The amino acid sequences of the first 10 amino acids of each of thefragments were compared with the known amino acid sequence for B.amyloliquefaciens NprE. This enabled the identification of the aminoacid at the N-termini and hence the cleavage site(s).

The generation of the citrate-induced fragments and their resolution wasshown on 10% SDS-PAGE. The sizes of the fragments were identified usinga standard molecular weight marker from Invitrogen. In the presence of10 mM citrate, two fragments in addition to remaining intact NprE wereobserved over the 100 minute time range. The two fragments formed at thelow citrate concentration were found to be 24 kDa and 9 kDa in size. Theintact nprE is 32 kDa. The 100-minute time range results in a goodproportion of cleaved protein (i.e., the primary autolysis fragments).No additional fragments were observed or detected under theseconditions. A study over 100 minutes in the presence of increasingcitrate was performed to obtain the secondary autolytic fragments. Inthis experiment, when concentrations between 10-30 mM citrate were used,the two fragments described above were observed. At 40 mM citrate, lessof the larger 24-kDa fragments were apparent however a 15-kDa fragmentwas also apparent. Between 50-100 mM citrate, the 24 kDa fragment andthe 9-kDa fragments were no longer detected but three other fragments,of sizes 21 kDa, 15 kDa and 11 kDa, were observed.

The identity of the N-termini of the 24 kDa, 9 kDa (first twofragments), and the 21 kDa, 15 kDa and 11 kDa (the next autolyticfragments) were determined using Edman degradation (Argo Bioanalytica).

TABLE 21 N-Terminal Sequence of Fragments Corresponding molecule SampleN-terminal Amino weight on Name acid sequence (5′-3′) SDS-PAGE (kDa)Band A1 AATTGTGTTL (SEQ ID NO: 215) 24 Band A2 DAGDYGGVHT(SEQ ID NO: 216) 9 AGDYGGVHTN (SEQ ID NO: 217) GDYGGVHTN(SEQ ID NO: 218) Band A3 AATTGTGTTL (SEQ ID NO: 219) 21 Band A4AATTGTGTTL (SEQ ID NO: 220) 15 Band A5 LSNPTKYGQP (SEQ ID NO: 221) 11

Bands A1, A3 and A4 have the native N-terminal sequence that matches theN-terminus for the intact NprE. The sequencing report for Band A2 showedthree fragments where the least intense sequence appeared to beidentical to the more intense sequence, except that it was two residuesand one residue shorter than the more intense sequences, respectively.This was consistent with a fraying of that particular protein fragment.The pattern and the sizes of the gel fragments suggest that the 15 kDa(Band A4) may be derived from the 21-kDa fragment (Band A3) and hencethe C-terminus is deduced to be at or near position 198. However, it isnot intended that the present invention be limited to this particularembodiment.

FIG. 35 provides the amino acid sequences for the various fragments (1-5or A1-5 for N-terminal sequencing purposes). Fragment 1 (A1) has theN-terminal residues equivalent to that for the intact native protein(SEQ ID NO:222), fragment 2 (Ad2) N-terminus starts at or near D220 (SEQID NO:223). The following two amino acid residues (A221 and G222) arealso highlighted because this fragment was identified as being frayed.Fragment 3 (A3) (SEQ ID NO:224) and fragment 4 (A4) (SEQ ID NO:225) havethe N-terminus of the intact protein, and fragment 5 (A5) (SEQ IDNO:226) starts at L198. The C-terminus of fragment 4 is likely to be ator near M138 (based on the size difference between A3 and A4). Thecorresponding fragment for A3 was not detected.

Trypsin digestion followed by LCQ-MS of the peptide maps for fragments 1through 5 positively identified several amino acid peptides within therespective fragments. These are highlighted in FIG. 35. The LCQ-MSprovided a positive control for the identity of the fragments.

Based on the N-terminal and LCQ-MS analysis of the cleavage fragments,primary cleavage sites were identified at amino acid positions D220,A221, G222, M138, and L198. These sites were targeted usingsite-directed mutagenesis and site-evaluation libraries of D220, A221,G222, L198, and M138, D139 were created. The mutant proteins werescreened for increasing stability in detergent and for BMI-washperformance, as indicated herein. In some instances, the amino acidsalongside these sites were also selected for protein engineering, inorder to ensure that the clip site was indeed targeted.

The protein engineering results clearly indicated that amino acidsubstitutions of either Pro or Glu at D220 generated an NprE moleculethat is more stable in detergent. In addition, additional stability wasafforded to the NprE molecule by replacing G222 with Cys, and M138 witheither Ile or Leu. In general, these specific amino acid substitutionsprovided the NprE with detergent stability advantages without theBMI-wash performance being compromised. Thus, these experiments provideimportant mapping data for the citrate-induced autolysis sites,facilitating the identification of key amino acid residues that alterand affect the overall stability of NprE. Citrate (a builder added todetergent matrices) destabilizes and autolyses NprE and is suggested todo so by chelating the essential calcium-bound atoms. The application ofNprE in extreme detergent conditions requires that a more stable NprEmolecule be used in these settings. In these experiments, substitutionsof one or more of the autolytic sites of NprE have resulted in a moredetergent-stable nprE molecule for use in these extreme detergents.

Example 22 Liquid Laundry Detergent Compositions

In this Example, various formulations for liquid laundry detergentcompositions are provided. The following liquid laundry detergentcompositions of the present invention are prepared:

Formulations Compound I II III IV V LAS 24.0 32.0 6.0 3.0 6.0NaC₁₆-C₁₇HSAS — — — 5.0 — C₁₂-C₁₅AE_(1.8)S — — 8.0 7.0 5.0 C₈-C₁₀ propyldimethyl 2.0 2.0 2.0 2.0 1.0 amine C₁₂-C₁₄ alkyl dimethyl — — — — 2.0amine oxide C₁₂-C₁₅ AS — — 17.0 — 8.0 CFAA — 5.0 4.0 4.0 3.0 C₁₂-C₁₄Fatty alcohol 12.0 6.0 1.0 1.0 1.0 ethoxylate C₁₂-C₁₈ Fatty acid 3.0 —4.0 2.0 3.0 Citric acid (anhydrous) 4.5 5.0 3.0 2.0 1.0 DETPMP — — 1.01.0 0.5 Monoethanolamine 5.0 5.0 5.0 5.0 2.0 Sodium hydroxide — — 2.51.0 1.5 1 N HCl aqueous #1 #1 — — — solution Propanediol 12.7 14.5 13.110. 8.0 Ethanol 1.8 2.4 4.7 5.4 1.0 DTPA 0.5 0.4 0.3 0.4 0.5 PectinLyase — — — 0.005 — Amylase 0.001 0.002 13 — Cellulase — — 0.0002 0.0001Lipase 0.1 — 0.1 — 0.1 nprE 0.05 0.3 — 0.5 0.2 PMN — — 0.08 — — ProteaseA — — — — 0.1 Aldose Oxidase — — 0.3 — 0.003 ZnCl2 0.1 0.05 0.05 0.050.02 Ca formate 0.05 0.07 0.05 0.06 0.07 DETBCHD — — 0.02 0.01 — SRP10.5 0.5 — 0.3 0.3 Boric acid — — — — 2.4 Sodium xylene sulfonate — — 3.0— — Sodium cumene — — — 0.3 0.5 sulfonate DC 3225C 1.0 1.0 1.0 1.0 1.02-butyl-octanol 0.03 0.04 0.04 0.03 0.03 Brightener 1 0.12 0.10 0.180.08 0.10 Balance to 100% perfume/dye and/or water #1: Add 1N HCl aq.soln to adjust the neat pH of the formula in the range from about 3 toabout 5.

The pH of Examples above 22(I)-(II) is about 5 to about 7, and of22(III)-(V) is about 7.5 to about 8.5.

Example 23 Hand Dish Liquid Detergent Compositions

In this Example, various hand dish liquid detergent formulations areprovided. The following hand dish liquid detergent compositions of thepresent invention:

Formulations Compound I II III IV V VI C₁₂-C₁₅AE_(1.8)S 30.0 28.0 25.0 —15.0 10.0 LAS — — — 5.0 15.0 12.0 Paraffin Sulfonate — — — 20.0 — —C₁₀-C₁₈ Alkyl 5.0 3.0 7.0 — — — Dimethyl Amine Oxide Betaine 3.0 — 1.03.0 1.0 — C₁₂ poly-OH fatty acid — — — 3.0 — 1.0 amide C₁₄ poly-OH fattyacid — 1.5 — — — — amide C₁₁E₉ 2.0 — 4.0 — — 20.0 DTPA — — — — 0.2 —Tri-sodium Citrate 0.25 — — 0.7 — — dihydrate Diamine 1.0 5.0 7.0 1.05.0 7.0 MgCl₂ 0.25 — — 1.0 — — nprE 0.02 0.01 — 0.01 — 0.05 PMN — — 0.03— 0.02 — Protease A — 0.01 — — — — Amylase 0.001 — — 0.002 — 0.001Aldose Oxidase 0.03 — 0.02 — 0.05 — Sodium Cumene — — — 2.0 1.5 3.0Sulphonate PAAC 0.01 0.01 0.02 — — — DETBCHD — — — 0.01 0.02 0.01Balance to 100% perfume/dye and/or water

The pH of Examples 23(I)-(VI) is about 8 to about 11

Example 24 Liquid Automatic Dishwashing Detergent Compositions

In this Example, various liquid automatic dishwashing detergentformulations are provided. The following hand dish liquid detergentcompositions of the present invention:

Formulations Compound I II III IV V STPP 16 16 18 16 16 PotassiumSulfate — 10 8 — 10 1,2 propanediol 6.0 0.5 2.0 6.0 0.5 Boric Acid — — —4.0 3.0 CaCl₂ dihydrate 0.04 0.04 0.04 0.04 0.04 Nonionic 0.5 0.5 0.50.5 0.5 nprE 0.1 0.03 — 0.03 — PMN — — 0.05 — 0.06 Protease B — — — 0.01— Amylase 0.02 — 0.02 0.02 — Aldose Oxidase — 0.15 0.02 — 0.01 GalactoseOxidase — — 0.01 — 0.01 PAAC 0.01 — — 0.01 — DETBCHD — 0.01 — — 0.01Balance to 100% perfume/dye and/or water

Example 25 Granular and/or Tablet Laundry Compositions

This Example provides various formulations for granular and/or tabletlaundry detergents. The following laundry compositions of presentinvention, which may be in the form of granules or tablet, are prepared.

Compound Formulations Base Product I II III IV V C₁₄-C₁₅AS or TAS 8.05.0 3.0 3.0 3.0 LAS 8.0 — 8.0 — 7.0 C₁₂-C₁₅AE₃S 0.5 2.0 1.0 — —C₁₂-C₁₅E₅ or E₃ 2.0 — 5.0 2.0 2.0 QAS — — — 1.0 1.0 Zeolite A 20.0 18.011.0 — 10.0 SKS-6 (dry add) — — 9.0 — — MA/AA 2.0 2.0 2.0 — — AA — — — —4.0 3Na Citrate 2H₂O — 2.0 — — — Citric Acid (Anhydrous) 2.0 — 1.5 2.0 —DTPA 0.2 0.2 — — — EDDS — — 0.5 0.1 — HEDP — — 0.2 0.1 — PB1 3.0 4.8 — —4.0 Percarbonate — — 3.8 5.2 — NOBS 1.9 — — — — NACA OBS — — 2.0 — —TAED 0.5 2.0 2.0 5.0 1.00 BB1 0.06 — 0.34 — 0.14 BB2 — 0.14 — 0.20 —Anhydrous Na Carbonate 15.0 18.0 — 15.0 15.0 Sulfate 5.0 12.0 5.0 17.03.0 Silicate — 1.0 — — 8.0 nprE 0.03 — 0.1 0.06 — PMN — 0.05 — — 0.1Protease B — 0.01 — — — Protease C — — — 0.01 — Lipase — 0.008 — — —Amylase 0.001 — — — 0.001 Cellulase — 0.0014 — — — Pectin Lyase 0.0010.001 0.001 0.001 0.001 Aldose Oxidase 0.03 — 0.05 — — PAAC — 0.01 — —0.05 Balance to 100% Moisture and/or Minors* *Perfume, dye,brightener/SRP1/Na carboxymethylcellulose/photobleach/MgSO₄/PVPVI/sudssuppressor/high molecular PEG/clay.

Example 26 Liquid Laundry Detergents

This Example provides various formulations for liquid laundrydetergents. The following liquid laundry detergent formulations of thepresent invention are prepared:

Formulations Compound I I II III IV V LAS 11.5 11.5 9.0 — 4.0 —C₁₂-C₁₅AE_(2.85)S — — 3.0 18.0 — 16.0 C₁₄-C₁₅E_(2.5) S 11.5 11.5 3.0 —16.0 — C₁₂-C₁₃E₉ — — 3.0 2.0 2.0 1.0 C₁₂-C₁₃E₇ 3.2 3.2 — — — — CFAA — —— 5.0 — 3.0 TPKFA 2.0 2.0 — 2.0 0.5 2.0 Citric Acid 3.2 3.2 0.5 1.2 2.01.2 (Anhydrous) Ca formate 0.1 0.1 0.06 0.1 — — Na formate 0.5 0.5 0.060.1 0.05 0.05 ZnCl2 0.1 0.05 0.06 0.03 0.05 0.05 Na Culmene 4.0 4.0 1.03.0 1.2 — Sulfonate Borate 0.6 0.6 1.5 — — — Na Hydroxide 6.0 6.0 2.03.5 4.0 3.0 Ethanol 2.0 2.0 1.0 4.0 4.0 3.0 1,2 Propanediol 3.0 3.0 2.08.0 8.0 5.0 Mono- 3.0 3.0 1.5 1.0 2.5 1.0 ethanolamine TEPAE 2.0 2.0 —1.0 1.0 1.0 nprE 0.03 0.05 — 0.03 — 0.02 PMN — — 0.01 — 0.08 — ProteaseA — — 0.01 — — — Lipase — — — 0.002 — — Amylase — — — — 0.002 —Cellulase — — — — — 0.0001 Pectin Lyase 0.005 0.005 — — — Aldose Oxidase0.05 — — 0.05 — 0.02 Galactose oxidase — 0.04 PAAC 0.03 0.03 0.02 — — —DETBCHD — — — 0.02 0.01 — SRP 1 0.2 0.2 — 0.1 — — DTPA — — — 0.3 — —PVNO — — — 0.3 — 0.2 Brightener 1 0.2 0.2 0.07 0.1 — — Silicone antifoam0.04 0.04 0.02 0.1 0.1 0.1 Balance to 100% perfume/dye and/or water

Example 27 High Density Dishwashing Detergents

This Example provides various formulations for high density dishwashingdetergents. The following compact high density dishwashing detergents ofthe present invention are prepared:

Formulations Compound I II III IV V VI STPP — 45.0 45.0 — — 40.0 3NaCitrate 17.0 — — 50.0 40.2 — 2H₂O Na Carbonate 17.5 14.0 20.0 — 8.0 33.6Bicarbonate — — — 26.0 — — Silicate 15.0 15.0 8.0 — 25.0 3.6Metasilicate 2.5 4.5 4.5 — — — PB1 — — 4.5 — — — PB4 — — — 5.0 — —Percarbonate — — — — — 4.8 BB1 — 0.1 0.1 — 0.5 — BB2 0.2 0.05 — 0.1 —0.6 Nonionic 2.0 1.5 1.5 3.0 1.9 5.9 HEDP 1.0 — — — — — DETPMP 0.6 — — —— — PAAC 0.03 0.05 0.02 — — — Paraffin 0.5 0.4 0.4 0.6 — — nprE 0.0720.053 — 0.026 — 0.01 PMN — — 0.053 — 0.059 — Protease B — — — — — 0.01Amylase 0.012 — 0.012 — 0.021 0.006 Lipase — 0.001 — 0.005 — — PectinLyase 0.001 0.001 0.001 — — — Aldose Oxidase 0.05 0.05 0.03 0.01 0.020.01 BTA 0.3 0.2 0.2 0.3 0.3 0.3 Poly- 6.0 — — — 4.0 0.9 carboxylatePerfume 0.2 0.1 0.1 0.2 0.2 0.2 Balance to 100% Moisture and/or Minors**Brightener/dye/SRP1/Nacarboxymethylcellulose/photobleach/MgSO₄/PVPVI/suds suppressor/highmolecular PEG/clay.

The pH of Examples 27(I) through (VI) is from about 9.6 to about 11.3.

Example 28 Tablet Detergent Compositions

This Example provides various tablet detergent formulations. Thefollowing tablet detergent compositions of the present invention areprepared by compression of a granular dishwashing detergent compositionat a pressure of 13 KN/cm² using a standard 12 head rotary press:

Formulations Compound I II III IV V VI VII VIII STPP — 48.8 44.7 38.2 —42.4 46.1 46.0 3Na Citrate 2H₂O 20.0 — — — 35.9 — — — Na Carbonate 20.05.0 14.0 15.4 8.0 23.0 20.0 — Silicate 15.0 14.8 15.0 12.6 23.4 2.9 4.34.2 Lipase 0.001 — 0.01 — 0.02 — — — Protease B 0.01 — — — — — — —Protease C — — — — — 0.01 — — nprE 0.01 0.08 — 0.04 — 0.023 — 0.05 PMN —— 0.05 — 0.052 — 0.023 — Amylase 0.012 0.012 0.012 — 0.015 — 0.017 0.002Pectin Lyase 0.005 — — 0.002 — — — — Aldose Oxidase — 0.03 — 0.02 0.02 —0.03 — PB1 — — 3.8 — 7.8 — — 4.5 Percarbonate 6.0 — — 6.0 — 5.0 — — BB10.2 — 0.5 — 0.3 0.2 — — BB2 — 0.2 — 0.5 — — 0.1 0.2 Nonionic 1.5 2.0 2.02.2 1.0 4.2 4.0 6.5 PAAC 0.01 0.01 0.02 — — — — — DETBCHD — — — 0.020.02 — — — TAED — — — — — 2.1 — 1.6 HEDP 1.0 — — 0.9 — 0.4 0.2 — DETPMP0.7 — — — — — — — Paraffin 0.4 0.5 0.5 0.5 — — 0.5 — BTA 0.2 0.3 0.3 0.30.3 0.3 0.3 — Polycarboxylate 4.0 — — — 4.9 0.6 0.8 — PEG 400-30,000 — —— — — 2.0 — 2.0 Glycerol — — — — — 0.4 — 0.5 Perfume — — — 0.05 0.2 0.20.2 0.2 Balance to 100% Moisture and/or Minors* *Brightener/SRP1/Nacarboxymethylcellulose/photobleach/MgSO₄/PVPVI/suds suppressor/highmolecular PEG/clay.

The pH of Examples 28(I) through 28(VII) is from about 10 to about 11.5;pH of 15(VIII) is from 8-10. The tablet weight of Examples 28(I) through28(VIII) is from about 20 grams to about 30 grams.

Example 29 Liquid Hard Surface Cleaning Detergents

This Example provides various formulations for liquid hard surfacecleaning detergents. The following liquid hard surface cleaningdetergent compositions of the present invention are prepared:

Formulations Compound I II III IV V VI VII C₉-C₁₁E₅ 2.4 1.9 2.5 2.5 2.52.4 2.5 C₁₂-C₁₄E₅ 3.6 2.9 2.5 2.5 2.5 3.6 2.5 C₇-C₉E₆ — — — — 8.0 — —C₁₂-C₁₄E₂₁ 1.0 0.8 4.0 2.0 2.0 1.0 2.0 LAS — — — 0.8 0.8 — 0.8 Sodiumculmene 1.5 2.6 — 1.5 1.5 1.5 1.5 sulfonate Isachem ® AS 0.6 0.6 — — —0.6 — Na₂CO₃ 0.6 0.13 0.6 0.1 0.2 0.6 0.2 3Na Citrate 2H₂O 0.5 0.56 0.50.6 0.75 0.5 0.75 NaOH 0.3 0.33 0.3 0.3 0.5 0.3 0.5 Fatty Acid 0.6 0.130.6 0.1 0.4 0.6 0.4 2-butyl octanol 0.3 0.3 — 0.3 0.3 0.3 0.3 PEGDME-2000 ® 0.4 — 0.3 0.35 0.5 — — PVP 0.3 0.4 0.6 0.3 0.5 — — MME PEG(2000) ® — — — — — 0.5 0.5 Jeffamine ® ED-2001 — 0.4 — — 0.5 — — PAAC —— — 0.03 0.03 0.03 — DETBCHD 0.03 0.05 0.05 — — — — nprE 0.07 — 0.080.03 — 0.01 0.04 PMN — 0.05 — — 0.06 — — Protease B — — — — — 0.01 —Amylase 0.12 0.01 0.01 — 0.02 — 0.01 Lipase — 0.001 — 0.005 — 0.005 —Pectin Lyase 0.001 — 0.001 — — — 0.002 ZnCl2 0.02 0.01 0.03 0.05 0.10.05 0.02 Calcium Formate 0.03 0.03 0.01 — — — — PB1 — 4.6 — 3.8 — — —Aldose Oxidase 0.05 — 0.03 — 0.02 0.02 0.05 Balance to 100% perfume/dyeand/or water

The pH of Examples 29(I) through (VII) is from about 7.4 to about 9.5.

While particular embodiments of the present invention have beenillustrated and described, it will be apparent to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

Having described the preferred embodiments of the present invention, itwill appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

Those of skill in the art readily appreciate that the present inventionis well adapted to carry out the objects and obtain the ends andadvantages mentioned, as well as those inherent therein. Thecompositions and methods described herein are representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It is readily apparent to oneskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims. The invention has beendescribed broadly and generically herein. Each of the narrower speciesand subgeneric groupings falling within the generic disclosure also formpart of the invention. This includes the generic description of theinvention with a proviso or negative limitation removing any subjectmatter from the genus, regardless of whether or not the excised materialis specifically recited herein.

We claim:
 1. An isolated variant Bacillus neutral metalloprotease havingat least 95% amino acid sequence identity to the parental polypeptide ofSEQ ID NO: 18, and comprising: (i) a substitution at least one of thepositions selected from the group consisting of S23, Q45, T59, S66,S129, F130, M138, V190, S199, D220, K211, and G222, with respect to theparental polypeptide, or (ii) a substitution selected from the groupconsisting of Q45E, T59P, S66E, S129I, S129V, F130L, M138I, V190I,S199E, D220P, D220E, K211V, K214Q, G222C, T004V/S023N, T059K/S66Q/S129I,T059R/S66N/S129I, and T059K/S66Q/S129V, wherein the variant has improvedthermostability compared to the parental polypeptide.
 2. The variantneutral metalloprotease of claim 1, wherein the substitution is selectedfrom the group consisting of S1291 and S129V.
 3. The variant neutralmetalloprotease of claim 1, wherein the substitution is selected fromthe group consisting of D220P and D220E.
 4. The variant neutralmetalloprotease of claim 1, wherein the Bacillus neutral metalloproteaseis a B. amyloliquefaciens neutral metalloprotease.
 5. The variantneutral metalloprotease of claim 1, wherein the thermostability of thevariant is at least 1° C. greater in a detergent composition compared tothe parental polypeptide.
 6. The variant neutral metalloprotease ofclaim 5, wherein the detergent composition is TIDE®.
 7. The variantneutral metalloprotease of claim 1, wherein thermostability isdetermined by differential scanning calorimetry at a concentration of220 or 440 ppm polypeptide.
 8. The variant neutral metalloprotease ofclaim 1, further having improved wash performance compared to theparental polypeptide.
 9. The variant neutral metalloprotease of claim 1,further having improved performance under lower or higher pH conditionscompared the parental polypeptide.
 10. The variant neutralmetalloprotease of claim 1, further having improved storage stabilitycompared the parental polypeptide.
 11. A cleaning composition comprisingthe variant neutral metalloprotease of claim 1.